C Streptomyces Gene Encoding 4 …shown that the initial events in melanin formation, involving the hydroxylation of L-tyrosine to L-DOPA(3-[3,4-dihydroxy-phenyl]-L-alanine) and its

Vol. 176, No. 17JOURNAL OF BACTERIOLOGY, Sept. 1994, p. 5312-53190021-9193/94/$04.00+0Copyright C 1994, American Society for Microbiology

A Streptomyces avermitilis Gene Encoding a 4-HydroxyphenylpyruvicAcid Dioxygenase-Like Protein That Directs the Production

of Homogentisic Acid and an OchronoticPigment in Escherichia coli

CLAUDIO D. DENOYA,* DEBORAH D. SKINNER, AND MARGARET R. MORGENSTERNBioprocess Research, Central Research Division, Pfizer Inc., Groton, Connecticut 06340

Received 3 May 1994/Accepted 22 June 1994

A 1.5-kb genomic fragment isolated from Streptomyces averninlis that directs the synthesis of a brownpigment in Eschenichia coli was characterized. Since pigment production in recombinant E. coli was enhancedby the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated withmelanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitiliswas unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototpe of melanin genes inStreptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single openreading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic andeukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressedin E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), whichis the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brownpigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients.Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations ofthis acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, theproduction of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitUis geneencoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated inthe medium by this clone.

The amino acid tyrosine can be either degraded to fumaricacid and acetoacetyl-coenzyme A or used in the biosynthesis ofmelanin (Fig. 1). In the oxidative degradation of tyrosine byvertebrates, it is first converted to 4-hydroxyphenylpyruvate bytransamination with alpha-ketoglutarate, in a reaction cata-lyzed by aromatic transaminase (30). 4-Hydroxyphenylpyru-vate dioxygenase (HPD), an enzyme present in most organ-isms, then catalyzes oxidation of 4-hydroxyphenylpyruvate to2,5-dihydroxyphenylacetate (homogentisic acid [HGA]) (Fig.1). This oxidation step is complex and involves hydroxylation ofthe phenyl ring and decarboxylation, oxidation, and migrationof the side chain (20). The enzyme has been purified fromhuman (33), chicken (51) and pig (40) livers and from aPseudomonas strain (34). The primary structures of Pseudomo-nas and mammalian HPDs were recently published (14, 41).Interestingly, the mammalian enzyme is approximately 90%identical in amino acid sequence to rat liver-specific alloanti-gen F (16, 18). F antigen is widely distributed among all groupsof vertebrates (37), but its biological function is not under-stood. In mammals, antigen F and HPD are expressed in theliver and kidney, have similar molecular masses, and probablyboth represent the same proteins (14). Recently, a Tetrahy-mena gene encoding a protein homologous to F antigen hasalso been reported (26).

Normally, HGA, the product of the reaction catalyzed byHPD, is further catabolized by the next enzyme in the pathway,homogentisate 1,2-dioxygenase (Fig. 1). In alkaptonuria, a rarehereditary metabolic disorder, the enzyme homogentisate 1,2-

* Corresponding author. Mailing address: Bioprocess Research,Pfizer Inc., Groton, CT 06340. Phone: (203) 441-4791. Fax: (203)441-3198.

dioxygenase is deficient (55). The urine of alkaptonuric pa-tients contains large amounts of HGA, which, on standing orwhen made alkaline and exposed to oxygen, turns dark becauseit is oxidized and polymerized to an ochronotic (derived fromochre; yellowish-brown color) pigment (29). Interestingly, for-mation of similar pigments resulting from the secretion andoxidation of either HGA or homoprotocatechuic acid (3,4-dihydroxyphenylacetic acid [DOPAC]) has been reported forsome bacteria (19, 50). In those cases, either DOPAC or HGAis probably produced through two alternative tyrosine cata-bolic pathways that use 4-hydroxyphenylacetate as an interme-diate (7, 9).

Tyrosine can also enter the oxidative pathway leading tomelanins (53), a class of polymeric pigments characterized bytheir intense dark color and lack of molecular regularity (13)(Fig. 1). Analysis of pigment production in many species hasshown that the initial events in melanin formation, involvingthe hydroxylation of L-tyrosine to L-DOPA (3-[3,4-dihydroxy-phenyl]-L-alanine) and its subsequent oxidation to dopaqui-none, are catalyzed by a single enzyme, tyrosinase (31). Allsubsequent oxidative polymerization steps leading from dopa-quinone to melanin are generally considered to occur sponta-neously through autoxidation (53). Melanin biosynthesis oc-curs in many species of higher organisms and bacteria,including many members of the genus Streptomyces (28). Thestreptomycete tyrosinase structural gene melC2 has beencloned from both Streptomyces antibioticus (27) and Streptomy-ces glaucescens (24), and their gene products have been shownto resemble tyrosinases of eukaryotes (36). Recently, a melClocus probably containing the tyrosinase structural gene wasalso found in Streptomyces michiganensis (22). Interestingly, agene directing the synthesis of a dark pigment in an Escherichia

5312

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S. AVERMITILIS HYDROXYPHENYLPYRUVATE DIOXYGENASE GENE 5313

HOyosH2CH(NHeCOOH

Tyrosine

To Catabolic Pathway

5 Tyrosinetransaminase

HOt3CH2-C-COOH

4-Hydroxyphenylpyruvic Acid(4-HPPA)

4-HPPA Dioxygenase(HPD)

CH2-COOH

HOoe HA

Homogentisic Acid (HGA)

2oGA1,2-Dioxygenase

To Biosynthesis of Melanins

Tyroslnase

HO QCH2CH(NHICOOH

HO

3,4-Dihydroxyphenylalanine(DOPA)

| Tyrosinase

0 CH2CH(NH2COOH

DOPAQ(pinone

Polymerization

Melanins

Fumaric Acid Oxidation,

Acetoacetic Acid Polymerization

Ochronotic Pigment

FIG. 1. Pathways of conversion of tyrosine to acetoacetic andfumaric acids and of biosynthesis of ochronotic and melanin pigments.The asterisk indicates the location of the enzymatic defect in alkap-

tonuria.

coli host was recently cloned from Shewanella colwelliana (17).The S. colwelliana gene encoded a protein that showed no

homology to any amino acid sequence in the databases. Thisgene was designated melA because it was presumed to mediatemelanogenesis via a novel mechanism (17).

Streptomyces avennitilis is an important industrial, gram-

positive, filamentous soil microorganism that produces eightdistinct but closely related antiparasitic polyketide compoundsnamed avermectins; S. avermitilis can also synthesize a mela-nin-like pigment (8). We recently reported the cloning of an S.avermitilis genomic DNA fragment that directs the synthesis ofa brown pigment when placed under the control of the lacpromoter in E. coli (49). Since the production of the brownpigment in the recombinant E. coli clone was enhanced by thepresence of tyrosine in the medium, it was inferred that thecloned DNA was involved in melanin biosynthesis via a

tyrosinase in S. avernitilis (Fig. 1).Here we report hybridization studies showing that the

pigment gene isolated from S. avermitilis is unrelated to thetyrosinase-encoding melC2 gene of S. antibioticus and S.

glaucescens (3). Nucleotide sequencing and computer-assistedhomology analyses of this genomic fragment revealed a singlegene encoding a protein with extensive homology to several4-hydroxyphenylpyruvate dioxygenases and F antigens (14, 18,

26, 41, 44) and to the protein encoded by the S. colwellianamelA gene (17). We also report the production of HGA and a

dark pigment similar to the ochronotic pigment observed in theurine of alkaptonuric patients by a recombinant E. coli clone inwhich the S. avermitilis structural gene for the HPD is overex-pressed. We conclude that the genetic determinant describedhere is indeed a gene encoding an HPD presumably involved inthe catabolism of tyrosine in S. avennitilis. The production ofochronotic-like pigment by heterologous expression in E. coli ispresumably due to the spontaneous oxidation and polymeriza-tion of HGA, the product of the reaction catalyzed by the S.avermitilis HPD, as in the urine from alkaptonuric patients.

MATERIALS AND METHODS

Microorganisms, plasmids, and growth conditions. S. livi-dans TK64 (generously provided by D. A. Hopwood, JohnInnes Institute, Norwich, United Kingdom), S. antibioticusATCC 11891, and S. avermitilis ATCC 31272 were grown inliquid YEME medium (25) at 29°C for 96 h, to preparechromosomal DNAs. pIJ702, a streptomycete high-copy-num-ber vector (27) carrying the S. antibioticus melC marker, wasalso provided by D. A. Hopwood. The components of the T7RNA polymerase-promoter system, namely, E. coli strainsDH1 and C600, harboring plasmids pT7-7 and pGP1-2, respec-tively, were kindly provided by S. Tabor (Harvard MedicalSchool, Boston, Mass.) and grown as previously described (48).pCD292 is a pGEM-3Z (Promega, Madison, Wis.) derivativecarrying a 1.5-kb EcoRV-SmaI S. avermitilis genomic fragmentinserted at the SmaI cloning site (49). E. coli DH5at competentcells were purchased from Life Technologies (Gaithersburg,Md.). E. coli cultures were grown in Luria-Bertani (LB)medium (42) supplemented, where indicated, with L-tyrosine(600 ,ug/ml) and/or ampicillin (50 ,ug/ml). In some experi-ments, to enhance pigment detection in the solid mediumsurrounding E. coli colonies grown overnight at 37°C, 1 ml of10 N NaOH was added on top of each 20-ml agar plate. Theplate (without the lid) was exposed to the air flow inside abiological hood for at least 1 h at room temperature.DNA isolation and manipulation. Total chromosomal DNA

from Streptomyces species was prepared by the cesium chloridegradient centrifugation procedure (25). Plasmids were isolatedfrom E. coli by a modification of the method of Birnboim andDoly (6), as described by Denoya et al. (11). General DNAmanipulations were performed as described previously (42).DNA probes were prepared by nick translation (42) by using[ct-32P]dClTP purchased from NEN (DuPont, Boston, Mass.)and the BRL Nick Translation System purchased from LifeTechnologies (Gaithersburg, Md.), following the instructionsobtained from the supplier. A cosmid library of S. avermitilischromosomal DNA, kindly provided by K. Stutzman-Engwalland E. L. McCormick, was prepared by using total genomicDNA that was partially digested with Sau3A and ligated intothe BamHI site in pKC505 (39), essentially as previouslydescribed (5).

Southern and colony hybridizations. DNA restriction frag-ments were transferred from 1% agarose gels to Nytranmembranes (pore size, 0.45 ,um; Schleicher & Schuell, Keene,N.H.) by the method of Southern (46). The genomic librarywas replicated onto sterilized nylon membranes, and colonieswere lysed and screened as previously described (5). The1.5-kb EcoRV-SmaI S. avermitilis cloned insert was isolated asan XbaI (site in vector)-SmaI fragment by electroelution froman agarose gel (42), labeled, and used to probe the cosmidlibrary. Three positively hybridizing cosmid clones (>2,200screened) were chosen and characterized by restriction map-ping. Hybridizations to [32P]DNA probes were carried out at42°C overnight in 50% formamide, 6x SSC (lx SSC is 0.15 M

\xidation

CH,-COOH

0 w0

Benzoquinoneacetic Acid

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NaCl plus 0.015 M sodium citrate) (42), 1ox Denhardt'sreagent (42), 1% sodium dodecyl sulfate (SDS), 100 ,ug ofdenatured, fragmented salmon sperm DNA per ml, and 100 ,ug

of E. coli tRNA per ml. The blots were washed twice with 1XSSC plus 0.1% SDS for 15 min each time at room temperatureand then twice with 0.1x SSC plus 0.1% SDS for 15 min eachtime at 42°C.DNA sequencing and computer analyses. The 1.5-kb EcoRV-

SmaI S. avermitilis DNA fragment (prepared from pCD292, as

before) was subcloned into SmaI-linearized bacteriophageM13mpl8 (35). Two white plaques from the mpl8 transfectioncarrying the insert in opposite orientations (CD440 andCD533) were selected. Nested sets of deletion mutants were

generated with exonuclease III by following procedures previ-ously described (23). The nucleotide sequence was determinedby the dideoxy sequencing method (43). DNA sequencing ofeach single-stranded DNA template was performed with theM13 '40 sequencing primer (New England Biolabs, Beverly,Mass.) and the TaqTrack sequencing kit (Promega) by follow-ing the instructions provided by the supplier and using 35S-dATP (NEN) to label the samples. DNA samples were run on

8% polyacrylamide-7 M urea gels prepared by following theinstructions provided with the Gel-Mix8 sequencing gel system

(Life Technologies). The Genetics Computer Group software(Madison, Wis.) (version 7.3) (12) was used for sequence

analysis. Deduced amino acid sequence data were comparedwith available databases (GenBank [release 80.0], EMBL[release 36.0], PIR-Protein [release 38.0], and SWISS-PROT[release 27.0]) by use of the FastA and TFastA programs, andcomparative alignments of amino acid sequpnces were ob-tained by using the Pileup and Pretty programs (12).

Construction and use ofE. coli expression plasmid pCD661.An NdeI restriction site spanning the ATG start codon was

introduced into the S. avermitilis pigment gene by PCR (42, 45)with pCD292 (see previous sections) as a template. DNAprimers (Genosys Biotechnologies, Inc., The Woodlands,Tex.) were 5'-AAATCTAGACATATGACGCAGACCACCCAC-3' (mutagenic, rightward) and 5'-AAGGATCCTGCAGCCCAGTCACGACGTTGTAAAACGA-3' (universal, left-ward). Reaction products were analyzed by electrophoresis ina 0.8% agarose gel. A PCR-amplified DNA fragment of thecorrect size (about 1.2 kb) was electroeluted, digested withNdeI and BamHI, and ligated into pT7-7 (48) to give expres-

sion plasmid pCD661, which was used to transform E. coliC600 carrying plasmid pGPl-2 (containing the T7 RNA poly-merase gene) (48). E. coli C600 pGP1-2/pCD661 and E. coliC600 pGP1-2/pT7-7 (control) were grown at 30°C in LBmedium containing 60 ,ug (each) of kanamycin and ampicillinper ml. When the cultures reached an optical density at 590 nmof 0.3 to 0.4, they were induced by raising the temperature to

420C for 30 min and then incubated at 370C for a further 90min. Uninduced control cultures were always kept at 30°C. Cellextracts were analyzed by SDS-polyacrylamide gel electro-phoresis (PAGE) as previously described (10).

Identification and quantitation of homogentisic acid byHPLC. The high-performance liquid chromatography (HPLC)system consisted of a ConstaMetric 3200 pump (ThermoSeparation Products, Riviera Beach, Fla.), a Gilson 232 Bioautosampler (Middleton, Wis.), and a Nucleosil C18 column (5,um; length, 200 mm; inside diameter, 4 mm; Macherey-Nagel,Duren, Germany). Substances were detected by a Spectro-monitor 5000 photodiode array detector (Thermo SeparationProducts) and chromatographic data were stored and calcu-lated with the Turbochrom version 3.3 interface software (PENelson, Cupertino, Calif.). Culture broth samples (1 ml) were

mixed with 100 ,ul of glacial acetic acid and clarified by

m E 3 fi -G E Emm00) I I I 1t I EI I I If

1 kb

FIG. 2. Restriction map of the region of the S. avermitilis genomethat contains the gene governing the production of a brown pigment inan E. coli host. Positions of restriction sites are indicated above theline. The black arrow below the map indicates the location andorientation of the ORF corresponding to the pigrnent gene.

centrifugation, and the recovered supernatants were stored at-20°C until assayed. Under these storage conditions, HGAconcentrations remained unchanged for at least 2 months.Frozen samples were thawed, diluted threefold with 10 mMacetic acid, filtered with a Millipore HV filter (pore size, 0.45,um; inside diameter, 13 mm), injected (20 i±l) on the Nucleosilcolumn, and eluted at a flow rate of 0.8 ml/min. The mobilephase was 10 mM acetic acid-methanol (85:15 [vol/vol]). Thewavelength was set at 292 nm, as previously described (1). Thepeak corresponding to HGA was identified and quantified bycomparing chromatograms of standard solutions of homogen-tisic acid (2,5-dihydroxyphenylacetic acid; Sigma, St. Louis,Mo.). The spectrum of HGA had an absorption maximum at290 nm. In addition, DOPAC and L-DOPA (both purchasedfrom Sigma) were used as internal standards in some experi-ments.

Nucleotide sequence accession number. The nucleotide se-quence reported here has been deposited at GenBank underaccession number U11864.

RESULTS

The S. avermitilis genomic sequence that directs productionof a brown pigment in E. coli is present in other streptomycetespecies and is not related to meiC. We previously described a

pGEM-3Z derivative (pCD292) carrying a 1.5-kb EcoRVSmaI genomic fragment from S. avermitilis (Fig. 2) thatimparted production of a brown pigment in an E. coli hostwhen the cloned function was under the control of the vectorlac promoter (49). The fact that the heterologous productionof pigment in E. coli was enhanced by addition of tyrosine tothe medium suggested that the S. avermitilis cloned sequencewas related to melanin biosynthesis (49, 53). We thereforedetermined if the S. avermitilis pigment fragment was relatedto melC2, the S. antibioticus gene which is considered theprototype of melanin-synthesizing genes in streptomycetes(27).

Southern blotting with the 32P-labeled 1.5-kb XbaI-SmaIinsert from pCD292 as a probe (Fig. 3A) showed that the S.avermitilis probe hybridized strongly, as expected, to the 2.3-kbBamHI S. avermitilis band from which it was cloned (49) andalso to single BamHI genomic fragments from S. lividans (9.0kb) and S. antibioticus (3.6 kb). Hybridization of the S.avermitilis probe to the 9.0-kb BamHI S. lividans fragment was

particularly intriguing, since S. lividans is naturally Mel- (25).In addition, this probe did not hybridize to any of the BclIlrestriction fragments of the streptomycete vector pIJ702,which includes a 1.6-kb BclI S. antibioticus genomic fragmentcontaining the melC2 tyrosinase structural gene (27).The same genomic DNAs were tested by using the melC2

probe. Figure 3B shows that the melC2 probe had a differenthybridization pattern, giving strong hybridization signals with a

4.7-kb BamHI genomic fragment from S. antibioticus and a

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S. AVERMITILIS HYDROXYPHENYLPYRUVATE DIOXYGENASE GENE 5315

A.1

(kb)

9.0 -

2 3 4

B.(kb)

1 2 3

7.4 -

4.73.6 -

2.3 -

FIG. 3. Detection of sequences homologous to the S. avermitilispigment producer gene and the S. antibioticus melC genes in variousStreptomyces species by Southern hybridization. Chromosomal DNA (4p,g) was digested with BamHI, and DNA fragments were electropho-resed in 1% agarose gels, transferred to nylon filters, and probed with32P-labeled 1.5-kb XbaI-SmaI DNA fragment from pCD292 containingthe S. avermitilis pigment determinant (A) or with 32P-labeled 1.6-kbBclI pIJ702 DNA containing the S. antibioticus melC genes (B) as a

probe. Species examined include S. lividans TK64, lanes 1; S. antibi-oticus ATCC 11891, lanes 2; and S. avermitilis ATCC 31272, lanes 3.pIJ702 digested with BclI was also included (lane 4 [A]). Fragments oflambda DNA cleaved with HindIII were used as size markers (rightmargin).

7.4-kb BamHI fragment (plus two additional fainter andsmaller bands) from S. avermitilis. As expected, there was no

hybridization of this probe to the Mel- S. lividans DNAfragments.These findings indicate that (i) the S. avermitilis pigment

sequence, which is located on a 2.3-kb BamHI genomicfragment (Fig. 2), is not homologous to the melC2 gene; (ii)sequences similar to those in the 2.3-kb BamHI S. avermitilisgenomic fragment are present also in other streptomycetespecies, including a Mel- strain; and (iii) S. avermitilis has anadditional BamHI fragment (7.4 kb) that strongly hybridizes tothe melC2 gene, suggesting that this fragment, and not the2.3-kb BamHI fragment discussed above, is related to melaninproduction in S. avernitilis.

Sequencing of the pigment region from S. avermitilis. Arestriction map of the chromosomal region containing thepigment determinant is shown in Fig. 2. The physical map ofthis chromosomal region was also confirmed by genomicSouthern analysis (data not shown). The complete EcoRV-SmaI fragment (1,469 bp) was sequenced. Its overall G+Ccontent was 68%. The sequence was analyzed for potentialcoding regions, using the CodonPreference program (12) andlooking for the characteristic codon usage and G+C third-position bias of Streptomyces genes (54). This analysis revealeda 1,143-nucleotide open reading frame (ORF) starting with a

potential ATG codon at position 312 and ending with a TAGstop codon at position 1452. A probable ribosome-binding site,GGAG, is located 14 nucleotides upstream of the start codonand shows a reasonable degree of complementarity to the 3'end of 16S rRNA from S. lividans (4, 47) and S. coelicolor (2).This ORF would encode a polypeptide of 380 amino acids withan estimated molecular mass of 41,653 Da.

Similarity of the deduced pigment gene product to HPD. Acomputer homology search of translated nucleotide and pep-tide sequence databases, using the deduced amino acid se-quence of the S. avermittis pigment gene, gave the surprisingresult that the best scores were several HPDs, including humanand porcine HPDs (14) (40 and 39% identity, respectively) andPseudomonas sp. HPD (41) (32% identity) (Fig. 4). A highsimilarity was also observed with rat and mouse liver-specificalloantigen F (18, 44) (44% identity) and to an F-antigen-likeprotein from Tetrahymenq thermophila (26) (42% identity).Finally, the S. avermitilis pigment gene product showed a highidentity (33%) to the deduced amino acid sequence of mel4, agene cloned from S. colwelliana (17) (Fig. 4). The amino acidsequence alignments in Fig. 4 suggest that the S. avermitilispigment determinant indeed encodes an HPD-like protein.Very likely, in the recombinant E. coli clone harboringpCD292, a protein that functions as a HPD that catalyzes theconversion of endogenous 4-hydroxyphenylpyruvate, an inter-mediate in the tyrosine catabolic pathway, to homogentisate,which is a precursor of ochronotic pigment, is produced. Thiscontention was substantiated by the experiments discussedbelow.

Overexpression of the S. avermnitilis pigment gene in E. coli.In pCD292, the putative pigment ORF translational startcodon was 0.3 kb downstream of the inducible lac promoter.To confirm the location of the translational start codon of thepigment gene and also to test the potential ORF and toimprove its expression, the S. avermitilis pigment gene wasoverexpressed in E. coli by using the T7 dual plasmid system(48). An expression plasmid was constructed for the S. aver-mitilis pigment gene by the strategy described in Materials andMethods. The mutagenic primer was designed to introduce anNdeI restriction site spanning the start codon of the pigmentgene and to change the third position of the fifth codon'(A toC) to reflect the most frequently used synonymous codonfound in 941 genes analyzed from E. coli (52). The PCR-amplified product was cloned into the NdeI and BamHI sites ofpT7-7 to obtain pCD661. This placed the pigment gene underthe inducible T7 promoter and in optimal position relative toa vector-encoded ribosome-binding site. E. coli C600 cellscontaining the plasmids pGP1-2, carrying the structural genefor T7 RNA polymerase under the control of a temperature-sensitive repressor, and pCD661 were analyzed for expressionby SDS-PAGE. Upon heat induction (42°C), a protein of about41 kDa, which is the approximate mass of the predicted S.avermitilis HPD protein, was detected in protein extracts fromC600 (pCD661) (Fig. 5). The 41-kDa protein was not found inthe vector-containing control strain.

Production of brown pigment and HGA in E. coli. While theclose homnology between the S. avermitilis gene described hereand the previously described HPD and F-antigen genesstrongly suggests that the former is indeed a HPD gene,confirmatory evidence was obtained by analyzing the charac-teristics of the pigment and by detecting the production ofHGA in the recombinant E. coli clone. Production of brownpigment by recombinant E. coli harboring pCD661 was ob-served after overnight culture at 37°C both on solid and inliquid media and was increased by the addition of tyrosine (Fig.6). This observation confirmed that the 41-kDa protein de-tected by SDS-PAGE in the induced strain harboring' PCR-derived construct pCD661 was responsible for producing thebrown pigment. Interestingly, pigment detection on solid me-dia was further increased by the addition of NaOH afterovernight incubation at 37°C. Furthermore, the pigment pro-duced in liquid culture was insoluble in 1 M NaOH and solublein 1 M HCI. These are characteristics of the ochronotic

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Sh MASEQNPPs ADLYENPSt WZQTTHH.....TPDTARQA DPFPVt ..GM DAVVVAVGNL KQAh.HYSTATe KSENKDHVVV GYTEKPVG.E RPTGGXFLGY DHLHIWVGK X9QUGWYTSRPi MTSYSDKGEK .......... .PERGRFLHF HSVTFWWVGA KQSi.SYYCSK

00 0 0 0 00 0 0000 0o 0 0

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Sh QNDINFLLNN EKQGFSALQFA KTEOPAISSM GWRVEDANFA FEG&VARCGK 100Ps QGAINLILNN EPHSVASYFA AINGPSVCGM AFRVKDSQKA YKRASZLQAQ 100St .......... PWGHFLADHV IEHGDGVVDL AIIVPD)RAL HAYk=HGAR 122

Te .......... DNQREMNQHBQ SLGDGVKDV AFAVEDCHSI YNKAIQRCAK 130

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0 0000 000000 00 0 0 00 0 0000000

Sh PAADEVKDLP Y......PAX YGIGDSLIYF IDTFGDDNNI YTSDFE ...A 141

Ps PIHIETGPME L.... NLPAI KGIGGAPLYL IDRFG..EGS SIYDIDFVFL 144St SVAEPYELXD EBGTVVLAAI A2YGKTRI!L VDRTGYDGPY LPGYV...A 169Te CAYPPQDLlD INGSVTIAAV HTYGEVIH!F IQRNDYKGFF MPGFVAHPLK 180Pi IVRLEVCCAA DVRGHHTPLD RARQVWEGTL VEKMTFCLDS RPQPSQTLLH 166

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Sh LDEPIITQEK GFIEVD8LTN NVHKGT)IEYW SNYKDIFGF T... .VRYFD 188Ps EGVDRHPVGA GLKIID8LTH NVYRGR8UYW ANFYEKLFNF R...ZIRYFD 191St APIVEPPAHR TFQAIDHCVG NVEUINEW VGrYNXVMY TNMKZFVGDD 219Te DPLNNVLPDI SYNYVDHIVCG NQPDNMMTSA ADWYEITLDF HRFWSVDDSM 230Pi RLLLSKLPKC GLEIIDHIVG NQPDQE)ESA SQWYMRNLQF HRFWSVDDTQ 216

00 0 000 000 00o00 0 00 0000000o 0 0 0

Sh IKGSQThLIS YALRSPDGSF CIPING.KG DDRNQIDBYI KEYDGPGVQH 237Ps IKGBYTGLTS KAMTAPDGMI RIPLNIE.SS XGAGQIEEFL MQFNGEGIQH 240St IATZYSALMS KCVVADGTLKV XFPINAPALA MSQIDZYL EFTGCQGVQH 269Te IHTIFSSLRS IVMTDYDQKI xmGNADG XRISQIQBYI DYYAGPGVQR 280Pi IHT1EYSALRS VVMANYEESI XMPIZIAPG IQSQIQZYV DYNGGAGVQN 266

000000 0 00000 0 0 000000 0 0000000000 0000000000

Sh LAFRSRDIVA SLDAMEGSSI QTLDIIPE.Y YDTIFLKL ......PQVTED 280

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Te IALNTSDVIN TVEGLRARCV EFLSI.PTBY YDNLRKAL.T AQTSITVKED 328Pi IILKTEDIIT AIRSIRERGV EFLAV.PFTY YKQLQFKLKS AK. .IRVKES 313

00000 oo 00 0000 00 000 0000*00* o 0 0000

Sh RDRIKHHQIL VD.GDBDC.. .!YLQnI!N L..FGPIFIZ IIQRKNNLGF 324Ps VGELOARGIL LDGSS1SGDK RLLLQIFSET L..MGPVFJZ FIQRKGDDGF 332St VDTLREIXIL AD..RDIDC.. YLLQIP VQD.PTVITZ IIERHCSMCF 358Te LDVLQKNHIL VD.YDEKG.. .ThLQIIr VEDRPTLIYZ IZQRNNHQCG 374Pi IDVLEILKIL VD.YDIKG.. YTLQDVS= MQDOREP2LZ VIQRNNHQGW 359

00 0 o00 0 o00 000000000 0000000000 00000 00

Sh GEGNI'ALIE SIZRDQVRRG VI........ .... 346 33% identity to St

Ps GEGKIOTA SIZRDQVRRB VLSTD......... 357 32% identity to St

St GKQNFKILIZ AlQgM NL...Z ..... .... 380Te GAGNYKSLFrV SLKLZQ1WaC NLTEIVKNIY .... 404 42% identity to StP1i GAGM SLFK EFIEEQZLRG NLTDTDPNGV PFRL 393 39% identity to St

0000oo0 o oo0oooo0 o0

FIG. 4. Alignment of the predicted amino acid sequence of the S.avermitilis HPD-like protein (St) with those of the S. colwelliana MelA(Sh) (17), Pseudomonas HPD (Ps) (41), T. thermophila F antigen (Te)(26), and porcine HPD (Pi) (14). Conserved positions are indicated asfollows: boldface, residues identical to those in the S. avermitilis geneproduct; 0, residues identical in all sequences; 0, residues eitheridentical or conservatively substituted in the S. avermitilis protein andat least two other proteins. The asterisks indicate Y and H residuesthat are conserved in all sequences and may be involved in metalbinding (41). Dots indicate gaps introduced to maximize alignment.

pigment produced by spontaneous oxidation and polymeriza-tion of HGA in the urine of alkaptonuric patients (29). Incontrast, melanins are readily soluble in alkali (53).To confirm that the product of the pigment ORF is indeed

an HPD, the production of HGA was analyzed in the fermen-tation cultures of E. coli harboring pT7-7 (vector) and pCD661(pigment ORF) by HPLC. Overnight cultures of the recombi-nant strain grown at 370C showed high levels of HGA, as

(KDa)

434-41

20-

FIG. 5. Synthesis of S. avermitilis HPD-like protein in E. coli C600examined by SDS-PAGE. Whole-cell extracts of E. coli C600 (pGP1-2)containing the expression vector pT7-7 (lane 2, uninduced, and lane 3,induced) and E. coli C600 (pGP1-2) containing the pT7-7 constructioncarrying the HPD-like gene (pCD661) (lane 4, uninduced, and lane 5,induced) were analyzed. The sizes of two prestained protein markers(ovalbumin and alpha-chymotrypsinogen) (lane 1) are indicated.

determined by the method of Akesson et al. (1), while thecontrol cells with the pT7-7 vector showed none. The peakshown in Fig. 7B at 5.85 min was identified as HGA, since itcomigrated with authentic standard HGA, and spiking theexperimental sample with pure HGA resulted in a single,larger peak without shoulders (not shown). Additionally, theUV spectrum of the 5.85-min peak present in the pCD661sample, as obtained with the photodiode array detector, wasidentical to that of the HGA standard. The small peak shownin the control sample (Fig. 7A) at approximately the sameretention time had a spectrum distinct from that of HGA (Fig.7A, inset), and presumably, it is composed of uncharacterizedbackground components. DOPAC and L-DOPA, two com-

A B

FIG. 6. Analysis of pigment production by transformants of E. coliDH5a harboring pCD661 (strain 661, top) or pT7-7 (strain 662,bottom) grown in LB plus ampicillin (A) or LB plus ampicillin plusL-tyrosine (B) plates overnight at 37°C.

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S. AVERMITILIS HYDROXYPHENYLPYRUVATE DIOXYGENASE GENE 5317

E

cn)

A

Retention Time (min)

cn

BI

15 V

Scan: 5.615 min14 0O.064

13

12

11210 250 30 n4

10

9

8

7

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Retention Time (min)

FIG. 7. HPLC chromatograms of culture broths. Samples

taken from the fermentation culture of theE. coli DH5a transformant

harboring pT7-7 (A) or pCD661 (B) after overnight growth at 370C

LB plus ampicillin liquid medium. Arrows indicate the 5.85-min

position which corresponds to the retention time exhibited by

HGA used as a standard when run under the same conditions

experimental samples. The spectra obtained from the 5.85-min peaks

from each sample are also shown (upper right corners).

pounds closely related to HGA, showed UV-absorbing peaks

well separated from HGA (9.20 and 2.60min, respectively).

DISCUSSION

Ochronosis is a rare condition in which the connective

tissues of alkaptonuric patients, particularly the cartilage,

sclera, and skin, become gradually darkened by the deposition

of a brown-to-black pigment. Ochronotic pigment, which

also observed in the urine of these patients, derives from the

spontaneous oxidation and polymerization of HGA. The first

step in the conversion of HGA to the ochronotic pigment

oxidation to the quinoid intermediate, benzoquinoneacetic

acid. The initial oxidation is thus analogous to the conversion

of dopa to dopaquinone (Fig. 1), the latter being an interme-

diate between dopa and dopachrome in the pathway of mela-

nin synthesis (53). It is generally believed that the autoxidation

and polymerization of benzoquinoneacetic acid to the ochro-

notic pigment resemble the mechanism by which dopaquinoneis converted into melanin (29). Similarly, oxidation of DOPACleads to the formation of a brown pigment having the appear-ance of both melanin and ochronotic pigment in Serratiamarcescens (50).Here we describe the sequencing and expression of an S.

avermitilis ORF encoding an HPD-like protein that directs thesynthesis of HGA in the E. coli host. HGA accumulates in thebroth of the E. coli recombinant clone harboring a vector onwhich the pigment ORF is efficiently expressed. Probably,HGA undergoes spontaneous autoxidation and polymeriza-tion, leading to the gradual production of a brown pigment onagar plates or in liquid cultures, a conversion that can beaccelerated by the addition of alkali. Thus, this process wouldbe analogous to the way HGA is converted to ochronoticpigment in the urine of alkaptonuric patients. Since addition oftyrosine to the culture medium enhances the production ofboth HGA and brown pigment in the E. coli clone, transami-nation of tyrosine to 4-hydroxyphenylpyruvate would beneeded to produce the substrate for the HPD-catalyzed reac-tion. There are three aminotransferases in E. coli capable ofconverting tyrosine into hydroxyphenylpyruvate, namely, theproducts of the tyrB, ilvE, and aspC genes (aromatic aminoacid, branched-chain amino acid, and aspartate aminotrans-ferases, respectively) (38). We speculate that tyrosine added tothe medium is catabolized via transamination by the E. colihost and that the 4-hydroxyphenylpyruvate produced in thisreaction is readily metabolized to HGA in the presence of thehigh concentration of HPD present, upon induction, in therecombinant clone. This leads to the accumulation of HGA inthe medium and subsequent autoxidation and polymerizationto ochronotic-like pigment. Since HGA can also be producedas a result of an alternative tyrosine catabolic pathway that uses4-hydroxyphenylacetate (4-HPA) as an intermediate (7), fur-ther studies will be needed to elucidate the substrate specificityof the S. avermitilis HPD. However, Hareland et al. (21) haveshown that HPD and 4-HPA 1-hydroxylase are two differentenzymes in Pseudomonas acidovorans and that the latteractivity is responsible for the conversion of 4-HPA to HGA.Recent comparative studies of the Pseudomonas, porcine,

and human HPD amino acid sequences showed that bothprokaryotic and eukaryotic forms of HPD are indeed membersof the same protein family (14,41). Database searching and themultiple alignment of amino acid sequences shown in Fig. 4revealed that the deduced structure of the S. avernitilis HPDpresented here exhibits a high degree of amino acid sequenceidentity to all the members of the HPD family, including therelated F-antigen sequences. The significant similarity pre-sented by the S. colwelliana melA gene product (17) and theproducts of the S. avermitilis pigment gene and other HPD andF-antigen genes (Fig. 4) deserves special consideration. Fuquaet al. (17) reported that the Shewanella gene directed thesynthesis of a dark pigment by anE.coli host, designating thisgene melA, because it was presumed to mediate melanogenesisvia an unknown mechanism. However, the alignments shown inFig. 4 strongly suggest that MelA is also an HPD-like protein,probably catalyzing, in the recombinantE. coli clone, theconversion of 4-hydroxyphenylpyruvate to HGA and the sub-sequent polymerization to ochronotic pigment, in a mannersimilar to that of the S. avermitilis gene product, reported here,in recombinantE.coli. Probably, and after reviewing the datain the light of the results presented here, a change of the nameof the previously reported melA gene of S. colwelliana would beappropriate.HPD belongs to the interesting group of intramolecular

dioxygenases, a class of enzymes catalyzing the incorporation

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5318 DENOYA ET AL.

of both atoms of molecular oxygen into a single substrate. Themechanism of this reaction is not fully known, but it ispresumed to include the formation of a peracid (oxene re-

agent), intramolecular transfer of one oxygen from the oxene

reagent to the aromatic ring, leading to the formation of a

benzene epoxide as an initial intermediate, and migration of analkyl group (20, 32). A transition metal, possibly iron, is likelyto be required in the formation of a peracid from the alpha-ketoacid and 02 in the reaction catalyzed by HPD. Recently,several tyrosine and histidine residues have been suggested as

potential metal-binding sites in the Pseudomonas HPD protein(41). Some of those residues appear to be highly conservedamong the HPD family members, including the S. avermitilissequence described here, as shown in Fig. 4.

Little information is available about the degradative path-way for many amino acids and other nitrogen-containingcompounds utilized by Streptomyces spp. and other gram-

positive bacteria (15). Although there is no evidence whetherthe HPD sequence reported here is directly involved in thecatabolism of tyrosine or in the production of an ochronotic-like pigment in S. avermitilis, further studies should contributeto a better understanding of the metabolism of aromatic aminoacids in these microorganisms.

ACKNOWLEDGMENTSWe thank D. A. Hopwood and H. A. I. McArthur for critical reading

of the manuscript. We also thank L. M. Collette and T. P. I for carryingout the HPLC analyses.

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