Pathways of Biosynthesis of Aromatic Acids and Vitamins ...biosynthesis generally, or on specific topics, are also available (12, 48). Therehas been, ofneces-sity, some selection in

BACnEIUOLOGICAL REVIEWS, Dec. 1968, p. 465-492Copyright © 1968 American Society for Microbiology

Vol. 32, No. 4, Pt. 2Printed in U.S.A.

Pathways of Biosynthesis of Aromatic AminoAcids and Vitamins and Their Control

in MicroorganismsFRANK GIBSON AND JAMES PITTARD

John Curtin School of Medical Research, Australian National University, Canberra, Australia, and School ofMicrobiology, University of Melbourne, Australia

INTRODUCTION................................................................ 465

INTERMEDIATES IN AROMATIC BIOsYNTHESIS ...................................... 466

Common Pathway........................................................... 466

Tryptophan Pathway........................................................ 468

Pathways to Phenylalanine and Tyrosine........................................ 469

Pathway to 4-Aminobenzoic Acid.............................................. 469

Intermediates in Ubiquinone Biosynthesis....................................... 470

Intermediates in Vitamin K Biosynthesis........................................ 471

Pathways Involving 2,3-Dihydroxybenzoate..................................... 472

Other Phenolic Growth Factors............................................... 473

ISOENZYMES AND PROTEIN AGGREGATES CONCERNED IN AROMATIC BiosYNTHESIS ........ 474Common Pathway........................................................... 474

Tryptophan Pathway......................................................... 474Phenylalanine and Tyrosine Pathways.......................................... 475

REGULATION OF THE COMMON PATHWAY ........................................ 476

Feedback Inhibition ofDAHP Synthetase....................................... 477

Repression ofDAHP Synthetase.............................................. 478

Inhibition of Other Enzymes of the Common Pathway............................ 480

Repression of Other Enzymes of the Common Pathway........................... 480

REGULATION OF THE TRYPTOPHAN PATHwAY ..................................... 481Feedback Inhibition.......................................................... 481

Repression................................................................. 481

REGULATION OF THE TYROSINE PATHWAY........................................ 482

Feedback Inhibition .......................................................... 482Repression................................................................. 482

REGULATION OF THE PHENYLALANINE PATHWAY .................................. 483

Feedback Inhibition.......................................................... 483

Repression................................................................. 483

REGULATION OF THE PATHWAYS OF VITAMIN BIOSYNTHESIS ............................ 483CHROMOSOMAL DISTRIBUTION OF GENES CONCERNED wrTH AROMATIC BIOSYNTHEsI s...... 484CONCLUSION.................................................................. 485

LITERATURE CITED ............................................................ 486

INTRODUCTIONThe aims ofthis review are to present an outline

of the metabolic pathways leading to the aromaticamino acids and vitamins and to discuss howthe flow of intermediates along these pathways iscontrolled. The general outlines of the pathwaysto the aromatic amino acids, phenylalanine,tyrosine, and tryptophan have been known forsome time, and they were excellently reviewedby Umbarger and Davis (162). Since then, thesituation regarding the "branch points" inaromatic biosynthesis has been clarified, andmuch information on the biochemical genetics

and control of the biosynthesis of aromaticamino acids has accumulated. In addition, thegeneral outlines of the pathways leading to themetabolically important compounds found insmall amounts, namely, 4-aminobenzoic acid,ubiquinone, vitamin K, and 2, 3-dihydroxy-benzoic acid, are partially understood. Thelatter compounds will be referred to as vitamins.

It is these more recent studies which we intendto emphasize with one important exception, thetryptophan operon. The biochemical genetics ofthis operon as a whole, and the enzyme trypto-phan synthetase in particular, have been studied

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intensively during recent years. The amount ofinformation now available on these topics war-rants a separate review; therefore, it is not ourintention to deal with this work in detail. Variousaspects of the work have been reviewed (171,172); other recent general reviews on aromaticbiosynthesis generally, or on specific topics, arealso available (12, 48). There has been, of neces-sity, some selection in the papers cited, butfurther references may readily be found throughthese.A general outline of the pathways to be dis-

cussed consists of a "common pathway" leadingthrough shikimate to chorismate, after whichthere is branching to the individual pathways(Fig. 1).

PHENYLAI

ERYTHROSE__ 4-PHOSPHATE

GLUCOSE +4 PHOSPHOENOL- "Common pathway"

PYRUVATE through shikimate

INTERMEDIATES IN AROMATIC BIosYNTHEsIs

Common Pathway

The common pathway involves the condensa-tion of two products of carbohydrate metabolism,phosphoenolpyruvate and erythrose 4-phosphate,to give a straight chain seven-carbon compoundwhich is then cyclized and undergoes a num-ber of reactions through shikimate to chorismate(Fig. 2).In recent work, the main advance has been the

clarification of the region of the branch point(Fig. 1) where, from chorismate, a series ofindividual pathways diverge. After the establish-ment of 3-enolpyruvylshikimate 5-phosphate asan intermediate on the common pathway (101,

LANINE TYROSINE TRYPTOPHAN

i CHORISMATE _ 4-AMINOBENZOATE __ FOLATE

2,3-DIHYDROXYBENZOATE UBIQUINONE VITAMIN K

2,3-DIHYDROXYBENZOYLSERINE

FIG. 1. General outline ofpathways for the formation ofaromatic amino acids and vitamins in E. coli.

PEP

COOH COOHI IC-O-P03H2 c=oIICH2 CH2 HO COOH-H2CHO I HO-C-H 2 3

3CH--OH I OHI H-C-OH

H-C-OH I OHI~rO-po3H2 H-C-OH OH

H-C-OH IH2

DHQCHr-O-po3H2 Ct-OH

NADPH

DHS SA

DAHPEP

COOH COOH COOHPEP CH -H3P04 CH

H OP-Ok )OH6 H2OP-0 0-C L O-tOH OH COOH OH COOKSAP EPSAP CA

FIG. 2. Intermediates in the common pathway of aromatic biosynthesis. Abbreviations: PEP, phosphoenolpyru-vate; EP, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; DHQ, 5-dehy-droquinic acid; DHS, 5-dehydroshikimic acid; SA, shikimic acid; SAP, shikimic acid 5-phosphate; EPSAP,3-enolpyruvylshikimic acid 5-phosphate; CA, chorismic acid. Trivial names of enzymes and some references topurification and cofactors:- (1), 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase;48, 154, 164); (2), 5-dehydroquinate synthetase (153); (3), dehydroshikimate reductase; (4), shikimate kinase;(5), 3-enolpyruvylshikimnate 5-phosphate synthetase (102); (6), chorismate synthetase (119).

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102), two groups studying the conversion ofshikimate to anthranilate showed that 3-enol-pyruvylshikimate 5-phosphate was a precursor ofanthranilate, as well as of phenylpyruvate and4-hydroxyphenylpyruvate (73, 138). It wassuggested that a specific branch point compoundwas involved, and this compound was sought byexamining a mutant in which the pathways totryptophan, tyrosine, and phenylalanine wereblocked (71, 72).

Using ultraviolet irradiation followed bypenicillin selection, a strain requiring bothtryptophan and tyrosine was isolated from atryptophan auxotroph which accumulated an-thranilate. The double mutant was then treatedto obtain the triple mutant (Aerobacter aerogenes62-1), in which tryptophan and tyrosine wereessential for growth, whereas phenylalaninestimulated growth. Cell-free extracts were pre-pared from this strain grown with excess trypto-phan to repress the enzyme system forminganthranilate. These cell extracts formed a newcompound from a mixture of shikimate, ribose-5-phosphate, adenosine triphosphate (ATP), andMge+. This compound could be converted toanthranilate in the presence of glutamine by cellextracts of a multiple aromatic auxotroph witha metabolic block immediately after 3-enol-pyruvylshikimate 5-phosphate. The new com-pound was readily isolated on paper chromato-grams and could be shown to be enzymicallyconverted not only to anthranilate but also toprephenate (and thence to phenylpyruvate and4-hydroxyphenylpyruvate), 4-hydroxybenzoate(at that time, a bacterial vitamin of unknownfunction), and 4-aminobenzoate (68, 71, 72).The new intermediate was named chorismic

acid (chorismic meaning separating) and foundto be excreted by whole cells of A. aerogenes62-1. It was isolated first as the barium salt andlater as the free acid, and its chemical structurewas determined (56, 66, 67, 69). Chorismic acidand its salts are unstable, and they decomposeunder physiological conditions to give a mixtureof 4-hydroxybenzoate and prephenate, thelatter compound giving phenylpyruvate in acidsolution (66, 72).

Chorismate, presumably because of a permea-bility barrier, does not act as a growth factor thatwill replace the amino acid or vitamin require-ments of multiple aromatic auxotrophs. Theinstability of chorismate at 37 C (66) necessitatesthe detection of any growth response during ashort period after its addition. Figure 3 showsthe results of an experiment in which the abilityof chorismate to substitute for the 4-amino-benzoate requirement of a multiple aromaticauxotroph (a mutant unable to carry out a

reaction of the common pathway) of Escherichiacoli was tested. The concentration of 4-amino-benzoate required for half-maximal growth ofsuch an auxotroph is about 10" M, but the addi-tion of a large excess of chorismate (5 X 10-4 M)did not support growth. The addition of dimethyl-sulfoxide (5%), which has been shown toincrease cellular permeability (61), did not affectthe results.The instability of chorismate and its inability

to promote growth probably were factors in thebranch point compound not being discoveredearlier. Metzenberg and Mitchell (115) examineda mutant of Neurospora crassa, which probablyaccumulated chorismate, in an attempt to find abranch point compound, but they found pre-phenate among other compounds.

Chorismate has also been isolated from culturefluids of E. coli (107), N. crassa (41), and Sac-charomyces cerevisiae (107), and it is also metabo-lized by cell extracts from Lactobacillus arabinosus(103), N. crassa (41), Claviceps paspalis (106),yeast (50, 104), and plants (27), indicating thegeneral role of the compound in aromatic bio-synthesis.

0.9

0.-

.0

450.7I-V

0.6

0.

4-Aminobenzoate

No addition

Addit Chorismate

0

6 8 10TIME (hr.)

12 14

FIG. 3. Inability of chorismate to replace the 4-aminobenzoate requirement of a multiple aromaticauxotroph (E. coli AB2839). Growth tests were carriedout as described previously (174). Medium containedmultiple aromatic supplement with 4-aminobenzoatelimiting growth at an absorbance ofabout 0.6. 4-Amino-benzoate (10-6 H) or chorismate (5 X 10-' m) wasadded at time indicated.

U.4 I IF mmmmmlml

9

I

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Early experiments on the incorporation of"4C-glucose into aromatic amino acids in A.aerogenes were not consistent with the scheme asoutlined, and they suggested that the shikimatering was not used, as such, as a precursor of thearomatic amino acids (for references, see 149).However, similar experiments have now beencarried out with E. coli (149), giving results whichare consistent with the pathway of aromaticbiosynthesis as it is now understood.

Tryptophan Pathway

No new intermediates in the tryptophan path-way have been found recently, and the pathwayis as set out in Fig. 4.The postulated intermediate, N-(5'-phos-

phoribosyl)-anthranilate, has not been isolatedand chemically characterized. Its existence hasbeen recognized by the enzymic formation of acompound with a lower intensity of fluorescencethan that of anthranilate by cell extracts of E. colt,A. aerogenes, Salmonella typhimurium, Sac-charomyces cerevisiae, and Pseudomonas aeru-ginosa (45, 50, 51, 57). The compound is verylabile (see 44), particularly under acid condi-tions, breaking down to regenerate anthranilate.Further evidence that the labile compound is anintermediate in tryptophan biosynthesis is pro-vided by the observation that it is formed by cellextracts from some tryptophan auxotrophs,whereas extracts from other tryptophan auxo-trophs will convert it to more stable compoundsfurther along the tryptophan pathway (51).

Because of the complexity of the reactioncatalyzed by anthranilate synthetase, inter-mediates between chorismate and anthranilatehave been proposed (102, 108, 136, 151). Theenzymic evidence suggests that a protein com-

GLUTAMIEWHO().COOH (.WMH) Mg l9

CHC-COOH A

CHORISMIC ACID

plex metabolizes chorismate through anthranilateto N-(5'-phosphoribosyl)-anthranilate. Evidencethat it is possible to trap an intermediate has beenobtained by Somerville and Elford (147), whofound that partially purified anthranilate syn-thetase from E. coli would catalyze the formationof a hydroxamate when incubated with cho-rismate, glutamine, and hydroxylamine. Althoughthe structure of the hydroxamate is not yetknown, the evidence obtained suggests that theoverall conversion of chorismate to anthranilatemay be separable into two steps, the first utilizingglutamine and the second being dependent onMg2+. Several possible intermediates have beentested, but they do not serve as precursors ofanthranilate (108, 151). Any mechanistic schemefor the amination of chorismate must take intoaccount the finding of Srinivasan (151) that theamide nitrogen of glutamine is transferred to thecarbon 2 of chorismate (Fig. 4). (The numberingof the carbons in compounds of the commonpathway is conventionally taken from the number-ing of shikimic acid, which is itself incorrectbecause the order ofnumbering should be throughthe double bond and not away from it.) Thisinformation was gained by growing a tryptophanauxotroph in a medium containing (3,4-14C)-glucose and determining the distribution of the'IC-labeled atoms in the excreted anthranilate.A comparison of the distribution of the "4C-labelin shikimate isolated from cultures of the ap-propriate mutant during earlier experiments withthe distribution of label in the 14C-anthranilateshowed the position of insertion of the nitrogenatom.The source of the nitrogen atom for anthra-

nilate formation has been the subject of a numberof studies. In the earlier experiments (150, 152),

tCOOH COOHPRPP [- r

NH2 2 NH-C-C-C-C-C-O -PO3H2H OH OH H H

APRA

coo*,IH. HHC CVHHHOPOHN4HO.I--C-C-6-0-PO3H2 41Y1NH) 6HA ) 1-.CH2-CH-CO

CDRP lP TRYPTOPHAN

FIG. 4. Intermediates in the tryptophan pathway. Abbreviations:- AA, anthranilic acid; PRA, N-(S'-phos-phoribosyl)-anthranilic acid; CDRP, I-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate; InGP, indoleglycerolphosphate. Trivial names ofenzymes and some recent references to purification and cactors:- (1), anrailaiesynthetase (123); (2), anthranilate-5'-phosphoribosyl-1-pyrophosphatephosphoribosyl transferase (PR transferase);(3), N-(5'-phosphoribosyl)-anthranilate isomerase (35); (4), indoleglycerol phosphate synthetase (35); (5), trypto-phan synthetase (34, 81, 169).

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it seemed that L-glutamine was the likely sourceof the nitrogen atom and that the amide nitrogenof glutamine was utilized (55, 152). A reexamina-tion of this problem (55) showed that, if bufferedat high pH, ammonium ions in relatively highconcentrations would serve as an effective nitro-gen source for anthranilate formation by crudecell extracts of A. aerogenes. Furthermore, it hasbeen possible to obtain evidence that ammoniumions may be used for anthranilate formationdirectly in vivo rather than via glutamine as anobligate intermediate (70). Thus, cell suspensionsof a double auxotroph that required tryptophanand were unable to form glutamine excretedanthranilate when incubated in a glucose-NH4+salts-buffer mixture.

Pathways to Phenylalanine and TyrosineThe intermediates between chorismate, phenyl-

alanine, and tyrosine, namely, prephenate,phenylpyruvate and 4-hydroxyphenylpyruvate(Fig. 5), have been known for a number of years(162). These intermediates are concerned in thephenylalanine and tyrosine pathways in E. coil,A. aerogenes, Saccharomyces cerevisiae, and N.crassa (8, 105, 162).Although no new intermediates have been

discovered, it has been found that the details ofthepathway in different organisms vary in the way inwhich the known intermediates are metabolized.

Pathway to 4-Aminobenzoic AcidThe molecule of folic acid (Fig. 6) contains a

benzene moiety which is inserted as 4-amino-benzoic acid (16); the biosynthesis of the lattercompound is being studied at present. Somemultiple aromatic auxotrophs were shown torequire 4-aminobenzoate for growth (38), andit seems that whether a requirement is shown by

COOH HOOC CH2-C-COOH

OH COOH OH

CHORISMIC ACID

such an auxotroph depends on the completenessof the metabolic block in the common pathway.'Leaky" mutants will allow sufficient flow ofintermediates along the common pathway tosatisfy the requirement for aromatic vitamins.Weiss and Srinivasan (166) showed that 4-amino-benzoate could be formed from shikimate 5-phosphate plus glutamine by cell-free extracts ofbakers' yeast. It was then shown (155) that theamide nitrogen of glutamine was the precursor ofthe amino group in the aromatic amine by using'5N-labeled glutamine and studying the effect ofglutamine analogues on the conversion.The fact that mutants blocked between 3-enol-

pyruvylshikimate 5-phosphate and chorismaterequired 4-aminobenzoate for growth in additionto the amino acids (40) indicated that chorismatemight well be a precursor of the bacterial vitamin.Cell-free extracts of the strain which accumulatedchorismate (A. aerogenes 62-1) were found toconvert chorismate to 4-aminobenzoate in thepresence of L-glutamine (68). Two differentapproaches to the problem of 4-aminobenzoatesynthesis are currently being used. Mutants ofboth N. crassa (52) and E. coil (83) requiring 4-aminobenzoate for growth have been divided intotwo classes by genetic mapping. Hendler andSrinivasan (80) reported "cross-feeding" betweenthe mutant strains of N. crassa, but no "cross-feeding" was found by Huang and Pittard (83)with the E. coil auxotrophs. The existence ofdifferent classes of mutants suggested that atleast two reactions were involved in the specificpathway for the synthesis of the vitamin, that is,between chorismate and 4-aminobenzoate. Moredirect biochemical evidence now supports thisconcept.

Cell extracts of yeast have been fractionatedby ammonium sulfate treatment yielding two

PHENYLPYRUVIC ACID PHENYLALANINECH2-C-COOH CHT-CH-COOH

3 NH2

PREPHENIC ACI 4CH2-C,-COOH CH2-CH-COOH

NADi:~i o transamination )

OH OH4-HYDROXYPHENYLPYRUVIC TYROSINE

ACID

FIG. 5. Intermediates in the biosynthesis ofphenylalanine and tyrosine. Trivial names of enzymes:- (1), choris-mate mutase; (2), prephenate dehydratase; (3), phenylalanine transaminase; (4), prephenate dehydrogenase; (5),tyrosine transaminase.

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fractions, neither of which alone forms 4-amino-benzoate from chorismate plus glutamine, butthe two fractions do so when mixed (80). Evidencefor an intermediate in E. coli has been obtainedby the use of cell extracts from suitable mutants(M. Huang, unpublished data). The two mutationsaffecting 4-aminobenzoate synthesis have beentransferred into a strain of E. coil K-12 unableto convert chorismate along the pathways to theamino acids. Cell extracts from the resultingstrains have been tested for their ability to convertchorismate into 4-aminobenzoate. Neither ex-tract alone will carry out the conversion, which is,however, carried out by a mixture of the twoextracts. The little that is known about 4-amino-benzoate synthesis in E. coil is shown in Fig. 7.

Intermediates in Ubiquinone BiosynthesisUbiquinone occurs in a wide variety of micro-

organisms and other cells (32). The structure ofubiquinone is shown in Fig. 8. The number ofisoprenoid units in the side chain varies with thespecies (32), although ubiquinones with varyingnumbers of isoprenoid units may be isolated

OH 1COOH

HONH~N~N*' LOH

FOLIC ACID

FiG. 6. Structure offolic acid.

COOH

r2CH2 X0

O C glutamineOH COOH

CHORISMIC ACID

COOH

NH2

4-AMINOBENZOICACID

FIG. 7. Biosynthesis of 4-aminobenzoic acid.

CH30ACH3 CH3

CH3O¾.0 CH2CH=CCH2 H

0

UBIQUINONEFIG. 8. Structure of ubiquinone.

from the one organism (64, 92). In E. coil andA. aerogenes, the ubiquinone with a forty-carbonatom side chain is the predominant form. We areconcerned here with the quinone nucleus ofubiquinone, which is derived from an aromaticprecursor.The observation of Rudney and Parson (139)

that "C-4-hydroxybenzaldehyde was incorporatedinto the benzoquinone ring of ubiquinone inRhodospirillum rubrum provided the first definiteevidence for an intermediate in ubiquinone bio-synthesis. 14C-4-Hydroxybenzoic acid, as wellas the aldehyde, was then shown to be incorpo-rated into ubiquinone in Azotobacter vinelandii,bakers' yeast and rat kidney (129) and R. rubrum(130). The relationship of ubiquinone to theshikimic acid pathway was shown by the demon-stration that 14C-shikimic acid was incorporatedinto ubiquinone in E. coil (28). In the latterexperiments, it was shown that an excess ofunlabeled 4-hydroxybenzoate in the mediumtogether with the "4C-shikimate "swamped" thelabeling of ubiquinone, providing further evidencethat 4-hydroxybenzoate lay on the pathway.

4-Hydroxybenzoate had been shown to havevitamin-like activity for multiple aromatic auxo-trophs of E. coil many years before (37), althoughthe requirement was not an absolute one. Multiplearomatic auxotrophs of E. coli growing on aglucose-mineral-salts medium supplemented withthe aromatic amino acids and 4-aminobenzoateformed ubiquinone only when 4-hydroxybenzoicacid was added (29). However, in similar experi-ments with multiple aromatic auxotrophs of A.aerogenes, ubiquinone was formed, suggestingthat an alternative pathway to ubiquinone mayexist in these cells. Experiments with cell-freeextracts of A. aerogenes (29; F. Gibson and R.Bayly, unpublished data) indicate 4-hydroxyben-zoate is formed from tyrosine (Fig. 9), althoughthe conversion of 4-hydroxyphenylpyruvate to4-hydroxybenzaldehyde occurs spontaneously atphysiological pH; it is not known whether thereis an enzyme carrying out this step (130). Underconditions where extracts of A. aerogenes readilyconvert 4-hydroxybenzaldehyde to 4-hydroxy-benzoate, extracts of E. coil K-12 are unable todo so, probably accounting for the inability ofthe multiple aromatic auxotrophs of the latterstrain to form ubiquinone, although there istyrosine in the medium.The pathway from 4-hydroxybenzoate to

ubiquinone is not well established, but the com-plete sequence shown in Fig. 10 has been proposed(63). A number of isoprenoid compounds havebeen isolated from cells of R. rubrum and P.ovalis (63, 65, 128, 129), and the plausible schemeof Fig. 10 has been advanced. However, it should

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-0-CHORISMATE ap PREPHENATE-*a 4-HYDROXYPHENYL-- 0 TYROSINE-PYRUVATE

4-HYDROXYBENZOATE

FIG. 9. Alternative pathways of4-hydroxybenzoate formation.

be emphasized that not all of the compounds setout in Fig. 10 have been isolated; furthermore,other related compounds not in the scheme havebeen isolated (65). The only reaction which hasbeen studied with cell-free extracts is the conver-sion of chorismate into 4-hydroxybenzoate (71).Recently, this reaction was studied in greaterdetail and the enzyme was partially purified(I. G. Young, unpublished data). The results ofinvestigation of other reactions of the proposedpathway, with cell-free enzymes, are awaited withinterest.Another approach to the problem of the bio-

synthesis of ubiquinone is to isolate mutantsunable to carry out specific reactions in the path-way and to look for accumulated precursors. Thisapproach has been used with E. coli K-12, butubiquinone does not give a growth response andis not required for growth in a glucose-mineral-salts medium. Hence, the usual methods ofmutant selection cannot be used. However, anindirect method of selection has been developed(31) which is based on the assumption thatubiquinone is essential for electron transport andthat, therefore, a ubiquinoneless strain of E.coit would grow fermentatively on a glucosemedium but be unable to grow on a reducedsubstrate such as malate or succinate as solesource of carbon. By testing strains of the desiredphenotype for their ability to form ubiquinone,a number of ubiquinoneless strains were isolated(31; G. B. Cox, unpublished data). One of thestrains contains two mutations affecting ubiqui-none biosynthesis (31). These mutations havebeen separated, by conjugation, into differentstrains. Examination of these strains for accumu-lated intermediates has shown that one of them(ubiA-) accumulates 4-hydroxybenzoate and theother (ubiBh) accumulates octaprenylphenol(G. B. Cox, unpublished data). The continuationof this approach should yield further informationabout the biosynthesis of ubiquinone.The methyl group of methionine has been

shown to serve as the source of the methoxy-methyl groups and the ring-methyl group ofubiquinone in Mycobacterium phlei and E. coil(90, 91).

COOH COOH

0-OcOH COOH OH

11

o ArCH30Q CH3 CH3CH30Ntr RH R= -(CH2CH= CCH2)-

x

FIG. 10. Proposed pathway of ubiquinone biosyn-thesis.

The pathway outlined above may be of generalsignificance since 14C-4-hydroxybenzoic acid isincorporated into ubiquinones in animal tissues(129), a plant (167), and a protozoan (118). Inthe last two cases, 'Cshikimate was also in-corporated into ubiquinone.

Intermediates in Vitamin K BiosynthesisAs in the case of the ubiquinones, a number of

forms of vitamin K occur in microbial cells. Theyhave a common naphthoquinone nucleus and aside chain which varies in the number of isopre-noid units and the degree of saturation and stereo-chemistry of the side chain (6, 32, 53, 86, 143).The basic structure of vitamin K is shown inFig. 11. One exception to this general structureis the 2-desmethyl nucleus in the vitamin Kisolated from Haemophilus influenzae (100).

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0

CH13HrCHH2 C=CCCH=CCH2+nH

0

VITAMIN K2FIG. 11. Structure of vitamin K2.

Virtually nothing was known about the bio-synthesis of the naphthoquinone nucleus ofvitamin K until 1964, when it was observed thatE. coil growing in the presence of 14C-shikimateincorporated the label into the naphthoquinonenucleus (28). It was later shown that the shikimatewas incorporated into the benzene ring of thenaphthoquinone (29). More recently, evidencehas been obtained that the carboxyl group ofshikimate is incorporated into vitamin K (19)and also into a plant naphthoquinone (177).

It is likely that the branch point from thecommon pathway for vitamin K biosynthesis isat chorismate, although the fact that chorismatedoes not act as a growth factor means that in-corporation of 14C-chorismate by the use of wholecells cannot be tested. However, a multiplearomatic auxotroph of A. aerogenes blockedbetween 3-enolpyruvylshikimate 5-phosphate andchorismate did not form vitamin K (29), and anexcess of unlabeled phenylpyruvate or 4-hydroxy-phenylpyruvate did not "swamp" the labeling ofvitamin K formed from 14C-shikimate. Thepossibility still remains that the branch point isat prephenate.The pathway from the branch point to vitamin

K is at present unknown. Two compounds, 3,4-dihydroxybenzaldehyde and a-naphthol, havebeen suggested as possible intermediates. In earlyexperiments, it was found that a compound withsome growth factor activity for multiple aromaticauxotrophs, 3,4-dihydroxybenzaldehyde (39),would "swamp" the incorporation of 14C-shikimate into vitamin K, although the effect wasnot as marked as the effect of 4-hydroxybenzoateon the incorporation of label into ubiquinone(28). Recently, it has been shown that 3H-3,4-dihydroxybenzaldehyde is not incorporated intovitamin K in E. coli, Bacillus subtilis, or M. phlei(19, 98), although the results of the swampingexperiment in E. coil were confirmed (98). a-Naphthol has been suggested as a precursor ofvitamin K (98), following the observation that

14C-a-naphthol is incorporated into vitamin K.Further evidence is needed, preferably with cell-free systems, to establish that a-naphthol isdirectly on the pathway of vitamin K biosynthesisand to clarify the effects observed with 3,4-dihydroxybenzaldehyde. No satisfactory systemfor the formation of the naphthoquinone nucleusofvitamin K by cell-free extracts has been devised,despite one promising report (4, 5).

It has been established for a number of organ-isms that the 2-methyl group of the quinonenucleus of vitamin K, like that of ubiquinone,is derived from methionine (4, 90, 91, 97).As in studies of ubiquinone biosynthesis, the

isolation of suitable mutants would assist in thesearch for possible intermediates. One mutantof E. coli K-12 unable to form vitamin K wasisolated during a search for ubiquinonelessmutants (31) and is being examined for possibleaccumulation products, but a rational procedurefor the isolation of mutants blocked in the specificpathway of biosynthesis of vitamin K has yet tobe devised.

Pathways Involving 2,3-DihydroxybenzoateThe importance of 2,3-dihydroxybenzoate in

bacterial metabolism was emphasized recentlywith the observation that it is an essential growthfactor for some multiple aromatic auxotrophs ofE. coli (30, 174). However, it has been known forsome time that 2,3-dihydroxybenzoate and cer-tain compounds containing the phenolic acidare formed by microbial cells. Ito and Neilands(88) isolated 2,3-dihydroxybenzoylglycine fromthe culture media of B. subtilis growing in aniron-deficient medium. Since then, 2, 3-dihydroxy-benzoate and related compounds have beenidentified as metabolic products formed by otherorganisms including A. aerogenes (132), C.paspali (3), Aspergillus niger (160), Streptomycesgriseus (54), S. rimosus (21) and E. coil (15, 174).In C. paspall (161) and Aspergillus niger (156),it appears that 2,3-dihydroxybenzoate may beformed from tryptophan, but in A. aerogenes andE. coli, it is formed more directly from chorismate.Crude cell extracts of A. aerogenes 62-1 (174) orof a similar mutant of E. coil K-12 (R. K. J. Luke,unpublished data) form 2,3-dihydroxybenzoatewhen incubated with chorismate, Mg2+ andnicotinamide mononucleotide (NAD). The path-way in A. aerogenes has been examined in detail(Fig. 12). Evidence for at least two steps in theconversion of chorismate to 2, 3-dihydroxy-benzoate was provided by the observation thatcrude extracts of A. aerogenes continued to form2,3-dihydroxybenzoate after all the chorismatehad been removed. When NAD was not added

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AROMATIC BIOSYNTHEESIS

COOH COOH COOH COOH

uH mH t -H H NAD )OHH H COOH COOHOHONl III lV

Fio. 12. Conversion of chorismate (I) to 2,3-dihydroxybenzoate (IV) through isochorismate (Ii) and 2,3-dihydro-2,3-dihydroxybenzoate (III).

CO-NH-CH2-COOH

,( OH

OH

CH20HCO-NH-CH-COOH

, OH

OH

2,3-DIHYDROXY- 2,3-DIHYDROXY-BENZOYLGLYCINE BENZOYLSERINE

FIG. 13. Structures of 2,3-dihydroxybenzoylglycine and 2,3-dihydroxybenzoylserine.

to the reaction mixture, chorismate was removedat about the same rate as when NAD was added.The compound formed by metabolism of choris-mate in the absence of NAD was isolated, ex-amined by nuclear magnetic resonance and massspectrometry (175; I. G. Young, unpublisheddata), and identified as 2,3-dihydro-2,3-di-hydroxybenzoic acid (Fig. 12).

Fractionation of crude extracts of A. aerogenes62-1 by chromatography on diethylaminoethyl(DEAE) cellulose gave a fraction which formedan intermediate capable of being converted to2,3-dihydro-2,3-dihydroxybenzoate and to 2,3-dihydroxybenzoate (I. G. Young, unpublisheddata). The new intermediate, for which the trivialname isochorismic acid is suggested, is veryunstable but has been isolated and identified(I. G. Young, T. J. Batterham, and F. Gibson,unpublished data) as the compound II of Fig. 12.The functional form of 2 ,3-dihydroxybenzoate

is not known. As mentioned above, 2,3-dihy-droxybenzoylglycine (Fig. 13) is formed by B.subtilis. A similar compound formed by E. coliwas tentatively identified as 2,3-dihydroxy-benzoylserine (15). The structure of the serineconjugate (Fig. 13) excreted by E. coil and A.aerogenes has recently been established by syn-thesis and by comparison with the natural product(I. G. O'Brien, unpublished data).

It has been observed that these compounds areformed in large quantities when cells are grown iniron-deficient media (14, 88; B. R. Byers and C. E.Lankford, Bacteriol. Proc., p. 43, 1967). Theenzymes forning 2,3-dihydroxybenzoate in A.

aerogenes (174) and the enzyme system converting2 ,3-dihydroxybenzoate to 2 ,3-dihydroxybenzoyl-serine in E. coli (14) are strongly repressed byiron or cobalt ions. Iron (or in one case, man-ganese ions) will replace the 2,3-dihydroxyben-zoate requirement of multiple aromatic auxo-trophs (174). These observations support thesuggestion of Ito and Neilands that the glycineconjugate might play an important role in irontransport (88). However, the effects of othermetals may mean that these phenolic compoundsare also important in the metabolism of metalsother than iron.

Mutants of E. coli K-12 requiring 2,3-dihy-droxybenzoylserine have been isolated to aid inthe study of the biosynthetic pathway and forstudies on function (R. K. J. Luke, unpublisheddata).

Other Phenolic Growth FactorsOther phenols have been found to act as growth

factors. Tyrosine or lower concentrations ofphenols such as protocatechuic acid or catecholacted as growth factors for a species of Sarcina(77) or Micrococcus lysodeikticus (140). Thepath-ways to these compounds are not known in theabove organisms; however, in N. crassa, protoca-techuic acid is formed from the common path-way intermediate, 5-dehydroshikimic acid (78).This compound has also been shown as the sourceof both protocatechuic acid and catechol in A.aerogenes by experiments with whole cells (133)and cell extracts (A. F. Egan, unpublished data).

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A species of Pseudomonas can convert anthran-ilate into catechol (157).

ISOENZYMES AND PROTEIN AGGREGATESCONCERNED IN AROMATIC

BIosymrsIs

Although not all of the reactions concernedin aromatic biosynthesis have been studied indetail, a number of interesting features have beenrevealed. These include the occurrence of iso-enzymes, protein aggregates carrying out morethan one reaction, and the catalysis of two reac-tions by one polypeptide. A brief survey of theoccurrence of these features in the various path-ways follows, and some aspects will be dealt within more detail when discussing metabolic regula-tion of the various pathways of biosynthesis.

Common Pathway

Isoenzymes have been found for three of thereactions of the common pathway. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthetase(DAHP synthetase), the first enzyme, has animportant function in the regulation of aromaticbiosynthesis and will be discussed in detail.Inhibitor studies, ammonium sulfate fractiona-tion, and column chromatography have providedevidence for the presence of isoenzymes of DAHPsynthetase (often three) in a wide variety ofmicroorganisms (17, 48, 49, 62, 93, 94, 145, 163,164), of which the best studied are E. coli andN. crassa. In B. subtilis, however, only a singleDAHP synthetase is present (96), as judgedby the results of enzyme purification, inhibitors,and the isolation of auxotrophic mutants lackingDAHP synthetase activity, which are the resultof single-step revertible mutations.Although multiple aromatic auxotrophs of

N. crassa that lack shikimate kinase activitymay be isolated (74), no similar mutants havebeen reported from the widely studied species

of E. coli, A. aerogenes, S. typhimurlum, and B.subtilis. The presence of two distinct shikimatekinases in S. typhimurium (120), and possiblyin B. subtifis (125), probably accounts for the lackof shikimate kinase mutants in this species, anda similar explanation may apply to the otherspecies. In N. crassa, however, no auxotrophslacking dehydroquinase were isolated, and againit was possible to demonstrate the presence oftwo enzymes (75); one of the enzymes was con-stitutive and the other was inducible.

In two organisms, B. subtilis and N. crassa,protein aggregates carrying out more than onereaction on the common pathway have beendescribed. A single revertible mutation in onestrain of B. subtifis, resulting in loss of chorismatemutase activity, gave a strain which simultane-ously lost DAHP synthetase activity. Gel filtrationof cell extracts suggested that one of the shikimatekinases might also be complexed with the twoenzymes mentioned (125). All the enzymes of thecommon pathway, with the exception of the first(DAHP synthetase) and the last (chorismatesynthetase), are associated as a multienzymecomplex in N. crassa (74).

Tryptophan PathwayOne of the best examples studied of a protein

aggregate is the terminal enzyme in tryptophanbiosynthesis in E. coli, tryptophan synthetase,which can be separated into two proteins, A andB (34, 171). Tryptophan synthetase from S.typhimurium is also dissociable, but that fromN. crassa is not dissociable, although it showsmany similarities to the E. coli enzyme (13).Although there are five recognizable reactionsin the specific pathway of tryptophan biosyn-thesis, it seems that in E. coll there are only threeenzymic steps, since anthranilate synthetase and5'-phosphoribosyl-1-pyrophosphate phosphori-bosyl (PR) transferase activities are associated

TABLE 1. Combination of PR transferase from A. aerogenes with anthranilate synthetase from E. coli

Anthranilate synthetase PR transferase

StrainaApprox molecular Inhibition by Approx molecular Inhibition by

weight tryptophan weight tryptophan

A. aerogenes (wild type) 170,000 + 170,000 +A. aerogenes NC3 Activity not detectable 90,000E. coli K-12 (wild type) 170,000 + 170,000 +A. aerogenes NC3, and E. coli 170,000 + 170,000 +D 9778b

a Anthranilate synthetase activity was not detectable in A. aerogenes NC3. No activity was detectablein E. coli D 9778.

b E. coli D 9778 was obtained from C. Yanofsky.

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in a protein complex (89), N-(5'-phosphoribosyl)-anthranilate isomerase and indoleglycerol phos-phate synthetase activities are carried by the samepolypeptide (35), and tryptophan synthetasecarries out the final step.

Anthranilate synthetase is evidently associatedwith PR transferase in E. coli (89) and S. typhi-muriwn (10, 11), since the two activities traveltogether during ultracentrifugation in sucrosegradients. Also, it was observed (89) that a non-sense mutation affecting the D gene coding forPR transferase activity in E. coli affected anthra-nilate synthetase activity, although the lattercould be detected on mixing extracts of such cellswith extract from cells in which the anthranilatesynthetase was affected by a mutation affectingthe E gene. Purification of the protein complexfrom E. coli results in loss of PR transferaseactivity because of instability (C. Yanofsky,personal communication). However, purificationof anthranilate synthetase from A. aerogenessimultaneously purifies the second activity (57;A. F. Egan, unpublished data). Furthermore, it ispossible to obtain evidence that anthranilatesynthetase from E. coli K-12, which is inactivealone, will combine with the PR transferase fromA. aerogenes to form an active complex (89;A. F. Egan, unpublished data). This evidence issummarized in Table 1. Thus, anthranilate syn-thetase and PR transferase in wild-type cells ofthese strains form a protein aggregate with amolecular weight of about 170,000, and bothreactions are inhibited by tryptophan. The mutantof A. aerogenes (NC3) lacking anthranilatesynthetase activity contains PR transferase activ-ity which has a lower molecular weight, as judgedby sucrose gradient centrifugation, than that inextracts from wild type, and it has lost its sensi-tivity to tryptophan. The mutant strain of E.coli used (D9778) has lost PR transferase activityand anthranilate synthetase activity owing to amutation in the gene coding for PR transferaseactivity (89). When crude cell extracts from themutants are incubated together and examined bycentrifugation in a sucrose gradient, there isfound a peak of anthranilate synthetase-PR

transferase activity with the molecular weight andsensitivity to tryptophan of the aggregate fromwild type A. aerogenes or E. coli.

It has recently been found that PR transferaseactivity is not necessary for anthranilate syn-thetase activity in P. putida and that anthranilatesynthetase itself in this organism is separable intotwo components (S. W. Queener and I. C.Gunsalus, Bacteriol. Proc., p. 136, 1968).

It can be seen that in E. coli (89) and S. typhi-murium (10) anthranilate synthetase activity isthe result of aggregation of two polypeptides.Similar combinations of polypeptides have beendemonstrated for enzymes of the tryptophanpathway in a variety of organisms, but the rela-tionships between the genes and the enzymesvary with the species. This point is well illustratedby reference to an extensive study by Hutter andDeMoss (84) of the biochemistry and geneticsof a variety of microorganisms, particularlyfungi.

Phenylalanine and Tyrosine PathwaysExamination of chorismate mutase activity in

A. aerogenes by chromatography of cell extractsshowed that there were two enzymes (24). Oneof these activities (chorismate mutase P) wasassociated with the next enzymic activity on thephenylalanine pathway, namely, prephenatedehydratase (see Fig. 5), and both activities wereabsent in a phenylalanine auxotroph. The proteincarrying out these activities was named the Pprotein. The second chorismate mutase (T) isassociated with prephenate dehydrogenase. Thisprotein aggregate from A. aerogenes has beenhighly purified (R. G. H. Cotton, unpublisheddata) and is dissociable into two subunits, neitherof which alone has either chorismate mutase orprephenate dehydrogenase activity (25, 26). Bothactivities of the T protein may be lost as theresult of a single revertible mutation (24). Thepathways in A. aerogenes 62-1 may be representedas in Fig. 14. The pathways in E. coli appear tobe the same as in Fig. 14, but a mutant has beenfound in E. coli that has lost prephenate de

[PREPHENATE] T PHENYLPYRUVATE - o PHENYLALANINE

CHORISMATE

[PREPHENATE]-.-* 4-HYDROXYPHENYLPYRUVATE -*- TYROSINE

Fio. 14. The pathways to phenylalanine and tyrosine in E. coli K-12 and A. aerogenes 62-1. There is a secondprephenate dehydratase in A. aerogenes 62-1.

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hydratase activity but still has two chorismatemutases (134).

In A. aerogenes 62-1, the strain which accumu-lates chorismate, there is a second prephenatedehydratase (A) which is still present in phenyl-alanine auxotrophs (28; Fig. 14). No specificfunction can be ascribed to this enzyme and it isnot present in the only other strain of A. aerogenesexamined (R. G. H. Cotton, unpublished data). A.aerogenes 62-1 has lost both T and P proteinactivities, but a reversion to tyrosine independencealso removes the phenylalanine requirement. Thesimplest explanation is that the prephenateformed by the T protein is then converted by theremaining prephenate dehydratase A into phenyl-pyruvic acid, thus bypassing the metabolic block.

Other organisms that have been examineddiffer from E. coil and A. aerogenes. In N. crassa(8) and the bean (Pisum sativum) (27), thereappears to be one chorismate mutase activitywith branching of the pathways at prephenate(as in Fig. 5). In extracts of one strain of B.subtilis, three distinct species (CM1, CM2, andCM3) of chorismate mutase could be separatedby chromatography on DEAE-cellulose, whereasextracts of another strain contained only the CM3species (109). Unlike the E. coil and A. aerogenessystem, there was no association of chorismatemutase with either of the subsequent activities.

REGULATION OF THE COMMON PATHWAY

Enzymes of the common pathway provide anintermediate, chorismic acid, which is a commonprecursor molecule of tyrosine, phenylalanine,tryptophan, folic acid, ubiquinone, vitamin K,and 2, 3-dihydroxybenzoylserine (or 2, 3-di-hydroxybenzoylglycine). Of these end-productsonly the three amino acids appear to play animportant role in controlling the rate of synthesisof chorismic acid. In different microorganisms,this control is affected either by feedback inhibi-tion alone or by a combination of feedbackinhibition and repression.

In feedback inhibition, it would be possiblefor the various end products to effectively controlthe common pathway by one of at least fourdifferent mechanisms. (i) In "cumulative inhibi-tion", as reported by Woolfolk and Stadtmanfor glutamine synthetase (170), each inhibitoradds its effect to the total inhibition of the enzyme,in which, however, the combined effect of anytwo inhibitors is less than the sum of their singleinhibitions. (ii) In "concerted or multivalentinhibition" (36), two end products are requiredtogether before any significant inhibition occurs.(iii) "Sequential feedback inhibition" is carriedout by a single molecule whose accumulation

TABLE 2. Percentage of inhibition of DAHPsynthetase isoenzymes of Escherichia coli

K-12 by the aromatic amino acidsa

DAHPTyrosine Phenylalanine Tryptophan

synthetase10-3 M 105 M 10-iM 10-5 MI 10-i-3 10-5 i

(Tyr) 95 50 5 0 0 0(Phe) 40 0 95 60 0 0(Trp)b 0 0 0 0 60 20

a Data taken from unpublished results of B. J.Wallace and J. Pittard. Single isoenzymes wereassayed in crude cell-free extracts obtained frommutant strains possessing only one functionalDAHP synthetase isoenzyme. The reaction mix-ture contained erythrose-4-phosphate (0.5 pmole),phosphoenolpyruvate (0.5 Mumole), sodium phos-phate buffer (pH 6.4; 25 umoles), a rate-limitingamount of enzyme and inhibitors at the final con-centrations shown above.

b Co2+ 10- M was added to the reaction mixture.

is in turn controlled by several end products(124). (iv) In "feedback inhibition of isoen-zymes," the reaction is carried out by more thana single enzyme, and a balanced control is possiblebecause an inhibitable isoenzyme exists for eachmajor end product. In this case, it is expectedthat the inhibition caused by two end productswill equal the sum of the inhibitions caused byeach one separately. If extensive cross-inhibitionsoccur, this result will not be obtained and the finaldistinction between (i) and (iv) may depend onthe physical separation of different isoenzymes.

Theoretically, similar possibilities exist for therepression of the formation of the enzymes ofthe common pathway. If there is no duplicationof enzymes, a system of multivalent repressioncould ensure that only in the presence of all theend products of the terminal pathways would theenzymes of the common pathway be repressed.Alternatively, if multiple enzymes are formedfor any particular reaction, repression of theformation of individual enzymes by individualend products offers a reasonably efficient systemof control.

Studies on the regulation of the common path-way of aromatic biosynthesis in a number ofdifferent microorganisms suggest that the controlof the first reaction of the pathway, the conversionof erythrose-4-phosphate and phosphoenolpyru-vate to DAHP, by inhibition or repression, orboth, is an important factor in the control ofthe common pathway. Therefore, this reactionwill be considered separately from the otherreactions of the common pathway. The control

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of this first reaction has also recently been re-viewed by Doy (48).

Feedback Inhibition ofDAHP SynthetaseSmith et al. (145) demonstrated the existence

of two DAHP synthetase isoenzymes in E. coli,DAHP synthetase (tyr) and DAHP synthetase(phe); DAHP synthetase (tyr) was inhibited bytyrosine and DAHP synthetase (phe) was in-hibited by phenylalanine. Brown and Doy (17),also working with E. coil W, demonstrated theexistence of a third isoenzyme, DAHP synthetase(trp), the activity of which was apparently notinhibited by either phenylalanine, tyrosine, ortryptophan, but the formation of which was re-pressed by tryptophan. The presence of threeisoenzymes in E. coil K-12 has been confirmedby the isolation of mutants which have lost oneor more of their isoenzymes, and by the identi-fication of these three activities using chroma-tography on DEAE-cellulose (163, 164; K. D.Brown and W. K. Maas, Federation Proc., p.338, 1966).With E. coil W, DAHP synthetase (phe) and

DAHP synthetase (tyr) activities have beenseparated by ammonium sulfate fractionationsof crude cell-free extracts (49). With E. coil K-12,recombinant strains have been isolated whichcontain only one of the three isoenzymes (164).In these strains, the activity of a single isoenzymecan be assayed in crude cell-free extracts. Inboth systems, the sensitivities of DAHP synthe-tase (tyr) and DAHP synthetase (phe) to inhibi-tion by phenylalanine, tyrosine, and tryptophanhave been determined (49). Similar results havebeen obtained with both organisms, and theresults of studies of inhibition with E. coil K-12are summarized in Table 2.There have been many reports to the effect that

DAHP synthetase (trp) from either E. coil Wor E. coil K-12 is not inhibited by tryptophan(49, 94, 107, 164). However, Doy has recentlyreported a 32% inhibition of this enzyme fromE. coil W (46). Because of the early difficultiesin establishing in vitro inhibition of DAHPsynthetase (trp), experiments have recently beencarried out to demonstrate inhibition of thisenzyme in whole cells (unpublished data). Whenthe trypR7 gene is present in a strain of E. coilK-12 which possesses only DAHP synthetase(trp), this enzyme and the enzymes of the trypto-phan operon are synthesized constitutively; i.e.,their rate of synthesis is no longer affected by thepresence or absence of tryptophan. These strainsrely entirely on DAHP synthetase (trp) to carryout the first reaction of the pathway, and theyhave a mean generation time in minimal medium

of 3 hr, compared with 80 min for the wild-typecells. When tryptophan is added to the medium,even though it has no effect on the formation ofDAHP synthetase (trp), the mean generationtime of these strains is increased to 12 hr. Whena further mutation which prevents the conversionof DAHP to dehydroquinate is introduced intothese strains, added tryptophan (5 X 10-4 M)reduces the rate of accumulation of DAHP by80%. The cells in both instances, however, con-tain the same levels of the enzyme DAHP synthe-tase (trp). Further studies with extracts haveshown that in the presence of Co2+ (10-3 M)DAHP synthetase (trp) is inhibited 60% by10-3 M L-tryptOphan (unpublished data).Using strains that each possess only one of the

isoenzymes, it has been possible to isolate feed-back-resistant mutants. The DAHP synthetase(trp) of one mutant isolated shows an inhibitionof 20% at 10-3 M L-tryptophan, compared with60% inhibition of the wild-type enzyme. TheDAHP synthetase (phe) of another feedbackresistant mutant shows an inhibition of 30% at10-4 M L-phenylalanine, compared with 92%for the wild-type enzyme (J. Pittard, unpublisheddata). Ezekiel has also reported the isolation ofmutant strains from E. coli K-12, in which DAHPsynthetase (phe) is no longer inhibited by phenyl-alanine (59). Although there have been no reportsyet of mutant strains in which DAHP synthetase(tyr) is feedback-resistant, there is no reason tobelieve that these should be difficult to isolate.

In S. typhimurium, the situation would appearto be very similar to that existing in the case ofE. coli (76). In crude cell-free extracts, inhibitionsby phenylalanine and tyrosine are found to beadditive when both amino acids are added to-gether, suggesting the presence of two separateisoenzymes. Furthermore, DAHP synthetase(tyr) and DAHP synthetase (phe), inhibitableby tyrosine and phenylalanine, respectively, canbe separated by ammonium sulfate fractiona-tion.

Evidence for the existence of a third isoenzyme,DAHP synthetase (trp), comes from the isolationof mutant strains unable to grow in minimalmedium supplemented with phenylalanine andtyrosine but able to grow in minimal medium.Mutant strains lacking DAHP synthetase (tyr)orDAHP synthetase (phe) have also been isolatedin this organism (76). No fraction of the DAHPsynthetase activity of S. typhimurium has yetbeen found to be inhibited by tryptophan (76,93). In view of the difficulty experienced in E. coli,however, this failure to demonstrate inhibitionin vitro may not reflect the true in vivo situation.A survey of the inhibition of DAHP synthetase

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from a variety of gram-negative and gram-positiveorganisms by phenylalanine, tyrosine, and trypto-phan has recently been carried out (94). In somecases (e.g., Hydrogenomonas sp.) phenylalanineand tyrosine exerted a cumulative feedbackinhibition on DAHP synthetase. In other organ-isms, the effects of the individual amino acids wereadditive. Therefore, it was concluded that eachamino acid was inhibiting a different isoenzyme.Many cases in which tryptophan could inhibitpart of the DAHP synthetase activity were de-scribed, and there were some strains whichappeared to possess only a single enzyme in-hibited by a single amino acid. As the authorspoint out, the dilemma posed by these last men-tioned strains, which are able to grow in thepresence of the amino acid which totally inhibitsDAHP synthetase in vitro, may well be resolvedwhen they are studied in more detail.

In Saccharomyces cerevisiae, results suggest(47, 105) that there are only two DAHP synthe-tase isoenzymes, one inhibited by tyrosine [DAHPsynthetase (tyr)] and one inhibited by phenyl-alanine [DAHP synthetase (phe)]. Mutants lack-ing either enzyme have also been isolated (117).The growth of these strains is inhibited by phenyl-alanine or tyrosine, respectively, confirming theexistence of only two isoenzymes. Furthermore,a recombinant strain which has neither isoenzymeis unable to grow on minimal medium andpossesses no detectable DAHP synthetase activ-ity (116).

In N. crassa, there are three isoenzymes, oneinhibited by tyrosine, one by phenylalanine, andone by tryptophan (46, 48, 93). One fraction,which can be isolated by chromatography onSephadex G-100, has been shown to be inhibited100% by tryptophan (93). Studies of the regula-tion of aromatic amino acid biosynthesis in C.Paspali reveal the presence of three isoenzymes106). One of these is inhibited by phenylalanine,sne by tyrosine, and one by tryptophan. In thisise the tryptophan-inhibitable isoenzyme con-

>titutes approximately 60% of the total activity.B. subtilis and a number of other strains of

Bacillus have a different means of inhibiting thefirst reaction of the pathway (95). In these strains,there appears to be only a single DAHP synthe-tase enzyme which is inhibited by either chorismicacid or prephenic acid. The accumulation ofchorismate and prephenate is, in turn, controlledby the amino acids phenylalanine, tyrosine, andtryptophan, acting on the reactions of the terminalpathways which utilize chorismate (124). Thesereactions will be dealt with in a later section. Inaddition to the various strains of Bacillus, strainsof Staphylococcus, Gaffkya, Flavobacterium,

Achromobacter, and Alcaligenes show sequentialfeedback inhibition of DAHP synthetase byeither prephenate or chorismate. The DAHPsynthetase from Xanthomonas, on the other hand,is inhibited approximately 86% by chorismate,but it shows 10% or less inhibition by prephenate(94).

Repression ofDAHP SynthetaseThe early work from two laboratories (17, 145)

established that in E. coli W there were threeDAHP synthetase isoenzymes and that theformation of each was repressed by a singleamino acid, e.g., DAHP synthetase (tyr) bytyrosine, DAHP synthetase (phe) by phenyl-alanine, and DAHP synthetase (trp) by trypto-phan. In addition, cross repression of DAHPsynthetase (tyr) by phenylalanine and tryptophanand DAHP synthetase (phe) by tryptophan wasalso reported (18). Recently, the derepressionof the DAHP synthetase isoenzymes has beenstudied by growing an aromatic auxotroph ofE. coli K 12 in a chemostat under conditions inwhich single aromatic amino acids, in turn.limit the growth rate (K. D. Brown, 1968.Genetics, in press). When either tryptophan orphenylalanine limits growth, DAHP synthetase(phe) is derepressed, and it is inferred from theseresults that phenylalanine and tryptophan to-gether are required for the repression of DAHPsynthetase (phe). DAHP synthetase (tyr) is onlyderepressed when tyrosine is the limiting aminoacid and when phenvlalanine and tryptophanare present at 104 M. If these last two amino acidsare present in higher concentrations (10-3 M),derepression of DAHP synthetase (tyr) is greatlyreduced. DAHP synthetase (trp), measured asDAHP synthetase activity not inhibited by eitherphenylalanine or tyrosine, is derepressed whenthe growth rate is limited by tryptophan (K. D.Brown. 1968. Genetics, in press). Studies carriedout on the repression of DAHP synthetase (tyr)in strains containing only this isoenzyme confirmthe finding that DAHP synthetase (tyr) is re-pressed in the presence of high concentrations ofphenylalanine and tryptophan (B. J. Wallace,unpublished data).

It has been known for some time that a muta-tion in a gene, trpR, can cause derepression ofthe enzymes of the tryptophan operon (23).Studies of trpR7 strains of E. coli K-12 whicheither possess all three DAHP synthetase iso-enzymes or possess only the single isoenzyme,DAHP synthetase (trp), have demonstrated thatDAHP synthetase (trp) is also produced con-stitutively in trpR7 strains (K. D. Brown. 1968.Genetics, in press).

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A second class of mutants has been isolated(unpublished data) in which the control of DAHPsynthetase (trp) has been altered. These mutantsalso make this enzyme constitutively but stillpossess a normally repressible tryptophan operon.Since the mutations conferring this change areclosely linked to the structural gene for DAHPsynthetase (trp), these mutants may turn out to beoperator constitutive mutants. In these mutants,DAHP synthetase (trp) is not only producedconstitutively, but is much less sensitive to in-hibition by tryptophan. This pattern resemblesthat of certain 5-methyl tryptophan-resistantmutants of E. coli possessing mutations in theanthranilate synthetase gene, as reported bySomerville and Yanofsky (148). Mutations in athird gene, the trpS gene, have an indirect effecton the levels of DAHP synthetase (trp) and theenzymes of the tryptophan operon; this will bediscussed later.The tyrosine analogues 4-aminophenylalanine

and 3-thianaphthenealanine have been found torepress the formation of DAHP synthetase(tyr), although they are not activated by tyrosyl-tRNA synthetase. Since these compounds canper se repress the formation of DAHP synthetase(tyr), it has been proposed that tyrosine uncom-plexed to any transfer RNA molecule should alsofunction as corepressor (137).

4-Aminophenylalanine at relatively low con-centrations (10-4 M) completely inhibits thegrowth of a recombinant strain of E. coil K-12possessing only DAHP synthetase (tyr). Since4-aminophenylalanine does not inhibit the activityof this enzyme (146), the inhibition of growth ispresumably caused by the repression of its forma-tion. Using this system, it is a simple matter toisolate mutant strains in which DAHP synthetase(tyr) is no longer repressed by tyrosine. Severalof these strains have been isolated, and one groupin particular has been studied in detail (B. J.Wallace and J. Pittard, J. Bacteriol., in press).In this case, a mutation in a gene designated astyrR, which is situated in the general region ofthe tryptophan operon (see Fig. 15), causes de-repression of DAHP synthetase (tyr), chorismatemutase T and its associated prephenate dehy-drogenase, and transaminase A. It also has aneffect on the repression of the shikimate kinaseenzyme. In other words, those enzymes normallyrepressible by tyrosine are made constitutivelyby these mutants. The sensitivity of DAHPsynthetase (tyr) to feedback inhibition by tyro-sine is, however, unchanged, as would be expectedfrom the fact that the tyrR gene and the aroFgene, the structural gene for DAHP synthetase(tyr), are widely separated on the chromosome.

TABLE 3. Repressed and derepressed levels of theDAHP synthetase isoenzymes in mutant strainsof E. coli K-12 possessing only a single DAHP

synthetase isoenzyme&

Specific activities in extractsprepared fromb

DAHP synthetase

Fully repressed cells Fully derepressedcells

(Tyr) 0 50(Phe) 11 18(Trp) 0.4 5-10

a Conditions under which the cells were grownare described in the text. Values obtained in thecase of strains possessing trpR- or tyrR- muta-tions are: DAHP synthetase (tyr) in a strainpossessing tyrR-, 150; DAHP synthetase (trp)in a strain possessing trpR7, 4.

b Specific activity = 0.1 Mmole ofDAHP formedper mg of protein per 20 min at 37 C.

In strains possessing the tyrRl mutation, highconcentrations of phenylalanine and tryptophanno longer repress DAHP synthetase (tyr), indi-cating that the gene product of the tyrR gene isinvolved in this apparent cross-repression (B. J.Wallace, unpublished data). No strains have yetbeen reported that are derepressed for the DAHPsynthetase (phe) isoenzyme.One interesting feature of the production of

each of the isoenzymes in E. coil K-12 and inE. coli W (K. D. Brown, Genetics, in press; 18)is to be found in the differences between fully re-pressed and fully derepressed values for eachone. In studies carried out in our laboratories,repressed values have been determined by grow-ing mutants containing single isoenzymes in thepresence of the three aromatic amino acids plusshikimic acid (10-6 M), to satisfy the requirementfor aromatic vitamins, and harvesting cells inlate exponential phase. Derepressed values havebeen obtained using strains containing only asingle isoenzyme, which were also unable to con-vert DAHP to 5-dehydroquinic acid (DHQ) be-cause of a mutation in the aroB gene. These weregrown, either in limiting shikimic acid or in amixture of the three amino acids in which therelevant amino acid was present in growth limit-ing concentrations. In the latter case, shikimicacid (10-6 M) was also added.The results of this study are indicated in Table

3. Two interesting points emerge from this table.The first is that under derepressed conditionsDAHP synthetase (tyr) activity is much higherthan that of either DAHP synthetase (phe) orDAHP synthetase (trp). The second point ofinterest is that the variation in levels of DAHP

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synthetase (phe) from repressed to derepressedstate is very small by comparison with DAHPsynthetase (tyr). Furthermore, although DAHPsynthetase (tyr) and DAHP synthetase (trp) arerepressed to very low values, DAHP synthetase(phe) exhibits much higher values for maximallyrepressed conditions.

Inhibition appears to play a much more im-portant role in the regulation of DAHP syn-thetase (phe) activity than does repression.Recombinant strains possessing only DAHPsynthetase (phe) are unable to grow in minimalmedium supplemented with phenylalanine orwith phenylalanine plus tryptophan. Mutantstrains, however, in which the DAHP synthetase(phe) is feedback-resistant suffer no reduction ingrowth rate when these amino acids are addedto the medium, even though the enzyme is asrepressible in these strains as in the parent (J.Pittard, unpublished data). Therefore, repressionby itself exerts little control on the in vivo activityof DAHP synthetase (phe). By contrast, however,4-aminophenylalanine, which acts only as a co-repressor, can completely inhibit growth of astrain possessing only DAHP synthetase [(tyr)B. J. Wallace and J. Pittard, J. Bacteriol., inpress], and a strain possessing only a feedback-resistant DAHP synthetase (trp) isoenzyme hasits growth rate halved by the addition of trypto-phan (J. Pittard and J. Camakaris, in prepara-tion).

In S. typhimurium, at least a 10-fold derepres-sion of the total DAHP synthetase activity occurswhen cells are transferred from medium con-taining excess phenylalanine and tyrosine to onein which these amino acids are present in verylow (1 pig/ml) concentration (76). However,whereas DAHP synthetase (tyr) is derepressedabout 10-fold in a "leaky" multiple aromaticauxotroph derived from S. typhimurium strainLT2, DAHP synthetase (phe) is not derepressed(189). In contrast, in a mutant strain of LT2resistant to ,B-2-thienylalanine a 12-fold de-repression of DAHP synthetase (phe) was ob-served (176). Other studies involving differentstrains of S. typhimurium indicate that when cellsare grown in minimal medium, DAHP synthetase(tyr) is the predominant isoenzyme, but bothDAHP synthetase (tyr) and DAHP synthetase(phe) can be derepressed about 10-fold by makingsuitable changes in the growth conditions (93).

In Saccharomyces cerevisiae, it has been re-ported that the formation of neither of the twoDAHP synthetases is repressed by phenylalanine,tyrosine, or tryptophan (47, 105).

In N. crassa, there appear to be three distinctDAHP synthetase isoenzymes. Although theirformation is not repressed by either tyrosine,

phenylalanine, or tryptophan to levels lower thanthose found in wild-type strains growing inminimal medium, derepression can be demon-strated by the use of auxotrophic strains, thusdemonstrating that some form of specific controldoes exist (48).

In B. subtilis, the formation of the single DAHPsynthetase enzyme is repressed by the aromaticamino acids (125), but no detailed studies of thisrepression have yet been reported.

Inhibition of Other Enzymes of theCommon Pathway

Studies of the control of a number of biosyn-thetic pathways have shown that when feedbackinhibition occurs, it almost always affects theenzyme carrying out the first reaction in a par-ticular biosynthetic sequence. Since the netresult of feedback inhibition is to stop the wastefulflow of intermediates along a pathway, it is notsurprising that the enzyme of the first reactionnormally functions as the major control point.There is currently only one reported case of

feedback inhibition of an enzyme of the commonpathway other than DAHP synthetase, and it isinteresting to note that in this case the affectedenzyme is found in close association with DAHPsynthetase. In B. subtilis, three enzymes, DAHPsynthetase, chorismate mutase, and shikimatekinase, form a protein aggregate (125). Theactivity of both DAHP synthetase and shikimatekinase is feedback-inhibited by both chorismateand prephenate.

Repression of Other Enzymes of theCommon Pathway

Relatively little work has been carried out onthe repressibility of the enzymes of the commonpathway other than DAHP synthetase. Studiesinvolving the growth of an aromatic auxotrophof E. coli K-12 in a chemostat under variousconditions failed to show any significant repres-sion or derepression of either dehydroquinatesynthetase or dehydroquinase (K. D. Brown.1968. Genetics, in press). Fewster (60) failed tofind any variation in the level of shikimate kinaseactivity in many strains of E. coli when grown inthe presence or absence of the aromatic aminoacids. Similarly, in S. typhimurium it has recentlybeen reported that the addition of excess aromaticamino acids to wild-type cells growing in mini-mal medium failed to repress any of the enzymesinvolved in converting DAHP to chorismate (76).

In contrast to these results, it has recently beenshown (J. Pittard et al., in preparation) thatunder certain conditions the shikimate kinaseactivity of E. coli K-12 can be considerably

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derepressed. When a wild-type strain is grown inthe presence of the aromatic amino acids, onlya twofold repression of this activity occurs, incomparison with extracts from cells grown inminimal medium. When, however, strains pos-sessing only DAHP synthetase (trp), which areeither trpR+ or trpR-, are grown in minimalmedium, the shikimate kinase activity is dere-pressed seven- to eightfold by comparison withfully repressed wild-type values. A similar resultis obtained when a strain which possesses thetyrRl mutation and has only DAHP synthetase(tyr) is grown in minimal medium. The additionof the aromatic amino acids to the minimal me-dium represses the formation of kinase activityin every case, although in strains containing eitherthe trpRl or the tyrR mutations, the fullyrepressed values are approximately double thoseobtained in the corresponding trpR+ and tyrR+strains. These results are summarized in Table 4.It can also be seen from Table 4 that when eithershikimic acid, tyrosine, or tryptophan limits thegrowth of an aromatic auxotroph, in contrast towhen these aromatic amino acids are present inexcess, a three- to fourfold derepression of thekinase activity occurs. When, however, phenylala-nine limits growth, there is no derepression ofkinase activity. Although these results do notindicate any simple system of control, they doclearly demonstrate that the levels of this par-ticular enzymic activity can be subject to con-siderable variations. Before these studies can beinterpreted in terms of any specific model, it isnecessary to establish whether the activity thatis measured in crude cell-free extracts representsone or more than one shikimate kinase enzyme.It has recently been demonstrated that in S.typhimurium there are two shikimate kinaseenzymes which can be separated from each otherby chromatography on DEAE-cellulose (120).

REGULATION OF THE TRYPTOPHAN PATHWAY

Feedback Inhibition

In E. coli (89), anthranilate synthetase and PRtransferase have been shown to form an enzymeaggregate such that, although the PR transferaseactivity can function by itself, only the aggregatehas appreciable anthranilate synthetase activity.Both anthranilate synthetase and PR trans-

ferase activity are inhibited by tryptophan.Feedback-resistant mutants have been obtainedby the use of tryptophan analogues such as5-methyl tryptophan. In a number of cases, thesestrains are found to be both feedback-resistantand to produce the enzymes of the tryptophanpathway constitutively (121, 148; C. Cordaroand E. Balbinder, Bacteriol. Proc., p. 51, 1967).

TABLE 4. A comparison of the specific activities ofshikimate kinase in different strains of E. coli

K-12 grown under different conditionsa

Specific activities in extractsprepared from cells grown in

Phenotype Minimal mediumMinimal plus phenylalanine,medium tyrosme, and tryp-

tophan(each, 10-3 m)

Wild type 3.4 1.6Multiple aromatic b 1.2cauxotroph

Prototroph possessing 12.0 2.5only DAHP synthetase(trp)

As above, but possessing 12.1 3.9the trpRt mutation

Prototroph possessing 4.9 1. 7conly DAHP synthetase(tyr)

As above, but possessing 10.7 3.0the tyrR- mutation

a Specific activity 0.1 Amole of substrateutilized per 20 min per mg of protein at 37 C.

b When the aromatic auxotroph was grown inlimiting shikimate, limiting tryptophan, limitingtyrosine, and limiting phenylalanine, respectively,specific activities were 3.5, 4.1, 5.4, and 0.5.

c 4-Aminobenzoic acid and 4-hydroxybenzoicacid (10-6 M) were also added to the medium.

These examples occur in E. coli K-12, E. coil W,and S. typhimurium. Only in the E. coli K-12mutants, however, has it been established thatboth changes were the result of a single mutation(148).In Chromobacterium violaceum (J. Wegman

and I. P. Crawford, Bacteriol. Proc., p. 115, 1967),P. putida (33), B. subtilis (124), Saccharomycescerevisiae (50, 105), and N. crassa (42), trypto-phan acts as a feedback inhibitor of anthranilatesynthetase, although in C. paspali (106), trypto-phan does not inhibit the activity of anthranilatesynthetase.

RepressionIn E. coli, the structural genes for the enzymes

of the tryptophan pathway are organized intothe now well-characterized tryptophan operon.Studies of various operator-constitutive mutantshave shown that the expression of all these genesis controlled by a single operator locus (112, 113,148). Furthermore, it has been shown (87) thatall the enzymes of the tryptophan pathway arerepressed or derepressed in a coordinate fashion.The regulation of these enzymes is controlled by

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the trpR gene, which was originally described byCohen and Jacob (23).

In the biosynthesis of at least two other aminoacids, histidine and valine (58, 122, 142), it hasbeen demonstrated that histidyl-transfer ribo-nucleic acid (tRNA) (his) and valyl-tRNA(val)are the active corepressors and not the aminoacids themselves. Consequently, mutations affect-ing tRNA molecules or amino acid activatingenzymes can also cause derepression of enzymesin a biosynthetic pathway. Furthermore, indiploids these mutations would be expected to berecessive in the same way in which a mutationwhich caused the formation of a nonfunctionalaporepressor is expected to be recessive. Hirstand DeMoss (Bacteriol. Proc., p. 114, 1967) havestudied the relationship between the size of thefree tryptophan pool and the repression oftryptophan synthetase in E. coil. They find thatchanges in this pool do not affect repression oftryptophan synthetase, and they conclude thateither there is more than a single intracellularpool of tryptophan or that tryptophan itself isnot the corepressor.A class of mutant strains which require trypto-

phan for growth has recently been described (43,82, 111) in which the mutations causing trypto-phan dependence map in the trpS gene, which islocated between the aroB and the pabA genes andfar away from the tryptophan operon. In spiteof the inability of these strains to grow withoutadded tryptophan, all of the enzymes of thetryptophan operon can be detected in their cell-free extracts. The levels of these enzymes inextracts prepared from derepressed trpS- cellsare, however, only one-third or less of the levelsobtained in extracts from derepressed trpS+cells (82). The level of DAHP synthetase (trp)is similarly lowered in trpS- strains (J Pittardand J. Camakaris, in preparation). Because ofthese observations, it seemed possible that thetrpS- strains may be producing a super-repressoranalogous to the il mutants of the lac operon(82, 111). Doolittle and Yanofsky (43), how-ever, have recently demonstrated that the trpSgene codes for the tryptophanyl-tRNA synthe-tase enzyme, and that trpS- mutants have agreatly reduced ability to charge tryptophan-specific tRNA. By contrast with the histidyl-tRNA synthetase mutants, in which the poorcharging of histidyl-tRNA causes derepressionof the histidine enzymes, the enzymes of thetryptophan pathway are not derepressed in thetryptophanyl-tRNA synthetase mutants. There-fore, it has been suggested that tryptophan, andnot tryptophanyl-tRNA, is the active corepressorfor the tryptophan operon (43). The low valueswhich have been observed for enzymes of the

tryptophan operon and for DAHP synthetase(trp) in trpS- strains is probably, therefore, adirect consequence of an internal accumulationof tryptophan by these strains.

In S. typhimurium, a single tryptophan operonexists, although it has been suggested that inthis organism the tryptophan operon containstwo separate promoter genes instead of one as inE. coli (10). Mutant strains resistant to 5-methyltryptophan and derepressed for enzymes of thetryptophan pathway have been isolated, but themutations have not yet been mapped (176).Preliminary studies in both Saccharomycescerevisiae and N. crassa (50) indicate that repres-sion plays an important role in these organisms.No repression of the tryptophan enzymes hasbeen found in C. paspali (106), and it has beenreported that tryptophan synthetase formation isspecifically induced in P. putida by indoleglycerol-phosphate (33).

REGULATION OF THE TYRosINE PATHWAYFeedback Inhibition

In A. aerogenes and E. coli, the first two reac-tions of the tyrosine pathway are carried out by asingle enzyme (24). Tyrosine is a feedback inhibi-tor of the second of these activities, prephenatedehydrogenase (90% inhibition at 10-3 M), but itdoes not affect the first, chorismate mutase T(24; B. J. Wallace, unpublished data). In strainsof B. subtilis, there are one or more chorismatemutase enzymes and a separate prephenate dehy-drogenase enzyme. Chorismate mutase is notinhibited by tyrosine, but this amino acid doesinhibit the prephenate dehydrogenase enzyme[90% at 103 M (124)].In S. cerevisiae, prephenate dehydrogenase

activity is activated by phenylalanine (105). InC. paspali, prephenate dehydrogenase is inhibitedby tyrosine (106).

RepressionIn A. aerogenes and E. coil, the formation of

chorismate mutase T and its associated prephe-nate dehydrogenase activity are strongly repressedby tyrosine (24). In E. coli, tyrosine has also beenshown to repress the formation of transaminaseA, an enzyme which converts 4-hydroxyphenyl-pyruvate to tyrosine (144). Mutations in a gene(tyrR) which is located at some distance on thechromosome from tyrA (the structural gene forchorismate mutase T) and its associated prephe-nate dehydrogenase cause constitutive synthesisof chorismate mutase T and prephenate dehy-drogenase, transaminase A, and DAHP syn-thetase [(tyr) B. J. Wallace and J. Pittard, inpreparation].

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AroF, the structural gene for DAHP syn-thetase (tyr), and tyrA are closely linked inthe E. coil chromosome and may be part ofan operon. The isolation of a mutant strainwhich has lost DAHP synthetase (tyr) activ-ity and which produces greatly reduced levelsof chorismate mutase T and prephenate de-hydrogenase (B. J. Wallace, unpublished data)lends support to this possibility. No operatorconstitutive mutants have yet been found. In S.cerevisiae (105), tyrosine does not repress prephe-nate dehydrogenase or chorismate mutase. Inthis strain, however, prephenate dehydrogenaseis induced by phenylalanine. The genetic unitsinvolved in this process of induction have notyet been studied.

REGULATION OF THE PHENYLALANINE PATHWAYFeedback Inhibition

In A. aerogenes and E. coli, phenylalaninefeedback inhibits prephenate dehydratase activity(90 to 100% inhibition at 103 M). Althoughphenylalanine has no effect on the associatedchorismate mutase P in E. coil (J. Pittard, un-published data), it causes 65% inhibition of thechorismate mutase P of A. aerogenes (24). InB. subtilis, phenylalanine inhibits prephenatedehydratase (124). Mutant strains of B. subtilishave been isolated that are resistant to fl-2-thienylalanine. In some of these mutants, phenyl-alanine activates prephenate dehydratase insteadof inhibiting it (22). In N. crassa and C. paspali,there appears to be a single chorismate mutaseenzyme. The activity of this enzyme is inhibitedby phenylalanine and by tyrosine, but the inhibi-tion is reversed and the enzyme is activated byL-tryptophan (7, 106). In C. paspail, phenyl-alanine also inhibits prephenate dehydratase activ-ity (106).

RepressionIn A. aerogenes and E. coli, phenylalanine

represses the formation of chorismate mutase Pand its associated prephenate dehydratase. Thevariation in activity between maximally repressedand derepressed levels is much less in the case ofchorismate mutase P than in that of chorismatemutase T (24; B. J. Wallace and J. Pittard, un-published data), and a transaminase enzymewhich is involved in the formation of phenylala-nine is found not to be repressed by phenylalanine(144). DAHP synthetase (phe) shows similarsmall variations in activity between maximallyrepressed and derepressed conditions. No regula-tor genes associated with the control of thispathway have yet been identified.

REGULATION OF THE PATHWAYS OF VITAMINBIoSYNwHEsIs

Although there is no doubt that the relativeamounts of any aromatic vitamin formed bybacterial cells can vary as a result of mutations orchanges in growth conditions, little informationis available concerning the mechanisms thatnormally control their synthesis. In part, thislack of information is due to the fact that thedetails of the pathways leading to the biosynthesisof the aromatic vitamins are still being workedout.Mutant strains of Staphylococcus aureus which

overproduce and excrete 4-aminobenzoic acidhave been reported (168). The formation of2,3-dihydroxybenzoate and related compoundshas been shown to be markedly influenced by themedium in which the cells are grown. Thus theamount of 2, 3-dihydroxybenzoate and 2,3-dihydroxybenzoylglycine produced in cultures ofB. subtilis is inversely proportional to the ironcontent of the growth medium (88). The forma-tion of enzymes concerned in the biosynthesis of2, 3-dihydroxybenzoate by A. aerogenes (174)and 2,3-dihydroxybenzoylserine by E. coll (14)is repressed by the presence of iron or cobalt inthe growth medium.The presence of the aromatic amino acids also

inhibits the production of 2, 3-dihydroxybenzoateand 3,4-dihydroxybenzoate by washed cellsuspensions of A. aerogenes (133). These effectsin A. aerogenes can be explained by feedbackinhibition of the DAHP synthetase system (A. F.Egan, unpublished data).

Vitamin K and ubiquinone levels are affectedby conditions of aeration (135), and in mutantswhich are unable to form one of the quinones,there is a several-fold increase in the level of theremaining quinone (31).There is no indication that the aromatic vita-

mins play any effective role in the control of thecommon pathway. On the other hand, since astrain of E. coil K-12 which has mutations in thestructural genes for DAHP synthetase (tyr),DAHP synthetase (phe), and DAHP synthetase(trp) possesses no detectable DAHP synthetaseactivity, there does not appear to be a fourthDAHP synthetase isoenzyme which is concernedwith vitamin biosynthesis. The results of thesein vitro tests are confirmed by the observationthat this same strain grows slowly with a meangeneration time of 280 min in a medium contain-ing phenylalanine, tyrosine, and tryptophan.When, however, either shikimic acid (106 M) or4-aminobenzoic acid, 4-hydroxybenzoic acid,and 2,3-dihydroxybenzoic acid (each at 10- M)are also added to the medium, the growth rate is

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returned to normal with a mean generation timeof 80 min (B. J. Wallace and J. Pittard, in prepara-tion).There are indications that not all of the DAHP

synthetase isoenzymes play an equal role invitamin biosynthesis. When a mutant strain of

trpR

FIG. 15. A map of the E. coli chromosome showingthe relative positions of genes concerned with aromaticbiosynthesis. The chromosomal locations of the genesubiA, ubiB, arol, and tyrR are based solely on the dataof interrupted mating experiments. The exact order ofthe genes pheA, aroF, tyrA and aroH, aroD has notbeen determined. Genes coding for enzymes of thecommon pathway have the prefix aro. Genes codingforenzymes of the tryptophan, phenylalanine, or tyrosinepathways have the prefixes trp, phe, and tyr, respec-tively. Genes concerned with the biosynthesis of ubiqui-none have the prefix ubi. Genes concerned with thebiosynthesis ofp-aminobenzoic acid have the prefix pab,and those concerned with the biosynthesis of 2,3-dihydroxybenzoic acid, dhb. Two genes concerned withregulation which affect both the common pathway andone of the terminal pathways have been given the prefixrelevant to the terminal pathway, e.g., trpR, tyrR. Theuppercase letters given to the genes have no significancewith regard to the relative positions of the enzymes inthe biosynthetic sequences. For example, aroA does notcode for the first enzyme of the common pathway. Thenumbers in parenthesis describe the particular reactionwith which the gene is concerned. For example, tyrA(1) codes for the first reaction in the terminal pathwayof tyrosine biosynthesis and aroF (1) t, codes for oneof the three isoenzymes involved in the first reaction ofthe common pathway. The subscript tyr denotes that itcodes for DAHP synthetase (tyr); aroG (M)ph. codesfor DAHP synthetase (phe) and aroH (1) trp codes forDAHP synthetase (trp). The function of arol is yet tobe determined. The gene trpS is the structural gene fortryptophanyl-tRNA synthetase. The formal representa-tion ofthe chromosome is in accordance with the recom-mendations of Taylor and Trotter (159).

E. coli containing only DAHP synthetase (tyr) isinoculated into minimal medium supplementedwith phenylalanine, tyrosine, and tryptophan,in the presence and absence of 10-6 M shikimate,its growth rate in the absence of shikimate isvery slow (mean generation time of 9 hr). In thepresence of 10-6 M shikimate, however, it growswith a mean generation time of 120 min. In astrain containing only DAHP synthetase (trp),the addition of shikimate to medium containingphenylalanine, tyrosine, and tryptophan causesonly a slight stimulation of growth rate, whereasin the strain containing DAHP synthetase (phe),this addition makes no difference to the growthrate. In medium not containing shikimate butcontaining phenylalanine, tyrosine, and trypto-phan, the mean generation times of strainscontaining only DAHP synthetase (trp) andDAHP synthetase (phe) are 114 and 130 min,r

Pathways of Biosynthesis of Aromatic Acids and Vitamins ...biosynthesis generally, or on specific topics, are also available (12, 48). Therehas been, ofneces-sity, some selection in

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