Biochemistry of coenzyme B 12 -dependent glycerol and diol dehydratases and organization of the encoding genes

Biochemistry of coenzyme B12-dependent glycerol and dioldehydratases and organization of the encoding genes

Rolf Daniel a;*, Thomas A. Bobik b, Gerhard Gottschalk a

a Institut fuër Mikrobiologie und Genetik der Georg-August-Universitaët, Grisebachstr. 8, D-37077 Goëttingen, Germanyb Department of Microbiology and Cell, Science Building 981, Room 1220, University of Florida, Gainesville, FL 32611, USA

Received 26 June 1998; received in revised form 19 August 1998; accepted 19 August 1998

Abstract

Glycerol and diol dehydratases exhibit a subunit composition of K2L2Q2 and contain coenzyme B12 in the base-on form. Thedehydratase reaction proceeds via a radical mechanism. The dehydratases are subject to reaction inactivation by the substrateglycerol which is caused by a cessation of the catalytic cycle because coenzyme B12 is not regenerated, instead5P-deoxyadenosine and a catalytically inactive cobalamin are formed. The genetic organization of the dehydratase genes isquite similar in all organisms. Downstream of the dehydratase genes an open reading frame encoding a polypeptide ofapproximately 600 amino acids was identified which is apparently involved in the reactivation of suicide-inactivatedenzyme. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Glycerol dehydratase; Diol dehydratase; Coenzyme B12 ; Glycerol utilization; 1,2-Propanediol degradation; dha regulon;

pdu operon

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5542. Distribution of glycerol and diol dehydratases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5543. Physiology and regulation of dehydratase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

3.1. Glycerol fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5543.2. 1,2-Propanediol fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

4. Reaction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5585. Assay systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5596. Biochemistry and molecular genetics of B12-dependent dehydratases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5627. Dehydratase in biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5638. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564

0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 2 1 - 7

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* Corresponding author. Tel. : +49 (551) 393827; Fax: +49 (551) 393793; E-mail: [email protected]

FEMS Microbiology Reviews 22 (1999) 553^566

1. Introduction

Glycerol dehydratase (glycerol hydro-lyase, EC4.2.1.30) and diol dehydratase (D,L-1,2-propanediolhydro-lyase, EC 4.2.1.28) can each catalyze the con-version of glycerol, 1,2-propanediol and 1,2-ethane-diol to the corresponding aldehydes [1,2]. These en-zymic reactions are known to proceed by a radicalmechanism involving coenzyme B12 as an essentialcofactor. Coenzyme B12 contains a unique covalentCo-C bond, which is stable in aqueous solution, buteasily undergoes homolytic cleavage in coenzymeB12-dependent enzymatic reactions. This homolyticcleavage is the initial step in the catalytic cycle ofall coenzyme B12-dependent enzymatic processes[3,4].

The coenzyme B12-dependent glycerol and diol de-hydratases are involved in the anaerobic utilizationof small molecules, they catalyze molecular rear-rangements that generate an aldehyde which can bereduced (i.e. glycerol fermentation) or dismutated tomore oxidized and more reduced compounds (i.e.1,2-propanediol fermentation). Coenzyme B12-con-taining ethanolamine ammonia-lyase of enteric bac-teria converts ethanolamine to acetaldehyde and am-monia, the aldehyde is proposed to dismutate toacetate and ethanol, reactions allowing the genera-tion of ATP in the acetate kinase reaction [5,6].Analogous reactions take place following the conver-sion of 1,2-propanediol to propionaldehyde by dioldehydratase [6^8]. The anaerobic degradation ofglycerol is initiated by two enzymes, glycerol dehy-drogenase forms dihydroxyacetone and glycerol de-hydratase yields 3-hydroxypropionaldehyde which isconverted further to 1,3-propanediol [1,9,10]. Glyc-erol dehydratases and the closely related diol dehy-dratases have been extensively studied in genera ofEnterobacteriaceae such as Klebsiella and Citro-bacter. It has been shown that these enzymes aresimilar in molecular masses and substrate spectra,but are di¡erent in monovalent cation selectivity pat-terns, a¤nity for coenzyme B12, and substrate spe-ci¢city [1,11^16].

2. Distribution of glycerol and diol dehydratases

The presence of glycerol and/or diol dehydratases

was shown in several Gram-positive and Gram-neg-ative microorganisms, including enteric and pro-pionic acid bacteria, solventogenic clostridia and lac-tobacilli [7,15,17^27]. All characterized dehydratasesare coenzyme B12-dependent, except the diol dehy-dratase of Clostridium glycolicum [28]. This enzymeseems to be entirely di¡erent from the B12-containingdehydratases and will not be discussed here any fur-ther. In the case of enteric bacteria, B12-dependentglycerol or diol dehydratases were detected in somestrains of the genera Salmonella, Klebsiella, Entero-bacter and Citrobacter [7,15,18,22,23,25,29]. Both de-hydratases may occur individually or together inthese organisms. The 1,2-propanediol- and non-glyc-erol-fermenting Salmonella typhimurium contain dioldehydratase exclusively. The glycerol-fermenting En-terobacter agglomerans, Klebsiella pneumoniae andCitrobacter freundii possess glycerol dehydratase,some strains of the latter two microorganisms alsopossess diol dehydratase. Growth on 1,2-propanediolinduces only the diol dehydratase, whereas both de-hydratases are present in anaerobically grown glyc-erol cells [15]. In one Klebsiella strain (ATCC 8724),the isofunctional diol dehydratase substitutes for thedefective glycerol dehydratase [2,16,22].

Glycerol dehydratase was detected in Clostridiumpasteurianum, Cl. butyricum, Lactobacillus sp., Lac-tobacillus reuteri, Lactobacillus brevis and Lactobacil-lus buchneri [19,20,24,27,30] and diol dehydratase inthe propionic acid bacterium Propionibacterium freu-denreichii [22].

3. Physiology and regulation of dehydrataseproduction

3.1. Glycerol fermentation

The pathway of glycerol breakdown and the keyenzymes and genes involved have been extensivelystudied in C. freundii and K. pneumoniae. Glycerolis converted by these bacteria to 1,3-propanediol(major product), ethanol, 2,3-butanediol, acetic andlactic acids [31]. In the absence of an external oxi-dant, glycerol is fermented by a dismutation processinvolving two pathways, one serving for glycerol ox-idation, the other for the consumption of reducingequivalents generated (Fig. 1A). Oxidation of glycer-

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R. Daniel et al. / FEMS Microbiology Reviews 22 (1999) 553^566554

ol is catalyzed by NAD�-linked glycerol dehydro-genase which converts the substrate to dihydroxy-acetone. This product is then phosphorylated by di-hydroxyacetone kinase and funneled into theglycolytic pathway [10]. Generation of NAD� isachieved by the NADH-linked 1,3-propanediol dehy-drogenase [32]. By the action of coenzyme B12-de-pendent glycerol dehydratase glycerol is ¢rst con-verted to 3-hydroxypropionaldehyde [1] which thenis reduced to 1,3-propanediol accounting for about50^66% of the glycerol consumed. All four key en-zymes and the corresponding genes have been iden-ti¢ed and characterized in C. freundii and K. pneumo-niae [1,10,18,21,32^34]. In C. freundii, the structuralgenes of the glycerol dehydratase (dhaBCE) are part

of the dha regulon along with the genes encoding thethree other key enzymes (dhaD, dhaK, dhaT) of thepathway (Fig. 1B). In addition, the dha regulon en-codes a transcriptional activator protein (DhaR)[10], a protein probably involved in reactivation ofglycerol dehydratase (OrfZ) [35], and three presump-tive proteins with unknown function (OrfW, OrfX,OrfY). The expression of the dha regulon is inducedunder anaerobic conditions when dihydroxyacetoneor glycerol are present. In contrast to the 1,3-prop-anediol-forming enteric bacteria, very little informa-tion is available about the genes and enzymes re-sponsible for glycerol utilization by clostridia andlactobacilli.

The glycerol fermentation pattern of clostridia is

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Fig. 1. Anaerobic utilization of glycerol. (A) Pathway used by enteric bacteria. (B) Apparent genetic organization of the C. freundii dharegulon. (C) Genetic organization of the reductive branch of glycerol utilization in Cl. pasteurianum. 1, dhaD, glycerol dehydrogenase;2, dhaK, dihydroxyacetone kinase; 3, dhaB, dhaC and dhaE, subunits of glycerol dehydratase; 4, dhaT, 1,3-propanediol dehydrogenase;dhaR, regulatory protein; orfW, orfX, orfY and orfZ, open reading frames with unknown function

R. Daniel et al. / FEMS Microbiology Reviews 22 (1999) 553^566 555

di¡erent, they form butyric acid and some strains ofCl. pasteurianum butanol and ethanol as additionalproducts ([17,30,36], for the entire pathway of sol-vent formation see [17]). All four key enzymes

known from the study of enteric bacteria were de-tected in crude extracts of Cl. pasteurianum [19] aswell as in Cl. butyricum, except that the dihydroxy-acetone kinase was not measured in the latter organ-

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Fig. 2. Anaerobic utilization of 1,2-propanediol. (A) Proposed degradative pathway. (B) Genetic and physical maps of the pdu locus of S.enterica LT2. The known promoter sites are indicated with the letter `P', and the direction of transcription is indicated by the arrow. ThepduGHJ genes have not yet been correlated to the physical map. ORFs 1^15 include homologs of the diol dehydratase reactivating pro-tein (DdrA) of K. oxytoca, alcohol dehydrogenase, acetate kinase, as well as four homologs of carboxysome shell protein genes.


ism [20]. These results indicate that clostridia fermentglycerol like the 1,3-propanediol-forming enteric bac-teria by a dismutation process. Recently, the genesencoding glycerol dehydratase and 1,3-propanedioldehydrogenase of Cl. pasteurianum were identi¢ed[19,37]. The deduced amino acid sequences and theproperties of the gene products are very similar tothose of 1,3-propanediol-forming enteric bacteria,but the genetic organization is di¡erent (Fig. 1B,C).In contrast to C. freundii and K. pneumoniae (notshown) the genes encoding glycerol dehydrataseand 1,3-propanediol dehydrogenase of Cl. pasteuria-num showed the same orientation and all pre-sumptive genes were located upstream of the dhaTgene.

The participation of glycerol dehydratase and 1,3-propanediol dehydrogenase in glycerol fermentationhas also been shown for some Lactobacillus speciessuch as L. reuteri, L. buchneri and L. brevis[24,26,27]. In contrast to the mentioned enteric bac-teria and clostridia, lactobacilli are not able to growon glycerol as sole carbon and energy source. Theseorganisms require a second substrate such as glucosefor glycerol utilization because of the absence of theenzymes for the oxidative branch.

3.2. 1,2-Propanediol fermentation

Diol dehydratase is used by some enteric and pro-pionic acid bacteria for the degradation of 1,2-pro-panediol [7,22]. Catabolism of this small molecule islikely to be important in a variety of nutritionallycomplex environments. 1,2-Propanediol is an end-product of the fermentation of rhamnose and fucose.These sugars are common in plant cell walls and arealso found in the glycoconjugates of mammalian in-testinal epithelial cells. Diol dehydratase catalyzesthe ¢rst step in the pathway of propanediol degra-dation, which produces propionaldehyde [38]. Subse-quently, propionaldehyde is converted to equalamounts of propanol and propionic acid. CoenzymeA-dependent aldehyde dehydrogenase, phospho-transacylase, propionate kinase, and alcohol dehy-drogenase are proposed to catalyze this dispropor-tionation (Fig. 2A) [7,8]. Fermentation of 1,2-propanediol provides one ATP per molecule of pro-panediol, but no source of carbon. Aerobically, 1,2-propanediol can provide both carbon and energy. It

is thought that the aerobic and anaerobic pathwaysare similar, but that oxygen allows the conversion ofsome propionyl-CoA to cell carbon.

In S. enterica (formerly called S. typhimurium) thestructural genes for diol dehydratase (pduCDE) arelocated in the pdu operon along with at least 20 addi-tional genes involved in propanediol degradation(Fig. 2B) [39,40]. The pdu operon encodes a pro-panediol di¡usion facilitator (PduF), a trans-criptional activator protein (PocR), and homologsof acetate kinase and alcohol dehydrogenase [39^41]. In addition, the pdu operon includes genes forthe reactivation of diol dehydratase, genes for theconversion of cobalamins to coenzyme B12, andgenes that are homologs of the shell proteins of car-boxysomes (polyhedral bodies that encase Rubiscoand which are thought to function in concentratingCO2) [40^42].

The apparent use of the carboxysome shell proteinhomologs is to encase diol dehydratase within a pol-yhedral shell. Thus far, four homologs of carboxy-some shell protein genes have been identi¢ed in thepdu operon [40]. Electron microscopy showed that S.enterica forms polyhedral bodies during growth on1,2-propanediol, and immuno-electron microscopyindicated that diol dehydratase was located withinthese polyhedra. The physiological reason for en-casement of diol dehydratase within these polyhedralshells is currently unknown. S. enterica is not anautotroph, it does not express Rubisco, and thereis no known role for CO2 in the degradation of1,2-propanediol.

Diol dehydratase is also subject to inactivation/re-activation. Inactivation occurs during catalysis withglycerol as substrate. Inactivation results from lossof the adenosyl group from the B12 cofactor [43].Reactivation is proposed to involve replacement ofthe inactive cofactor with coenzyme B12. Work withK. oxytoca has shown that two genes ddrA and ddrBare involved in reactivation [44]. A homolog of ddrA(orf1) is found in the pdu operon.

In S. enterica, expression of diol dehydratase isregulated via transcriptional control of the pdu oper-on. Both 1,2-propanediol and poor growth condi-tions are required for high expression. In addition,induction of the pdu operon is coordinated with in-duction of adjacent cobalamin biosynthesis (cob) op-eron [45]. Co-expression of the pdu and cob operons

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is achieved through the PocR, an activator proteinwhich mediates induction of both operons in re-sponse to propanediol. Two global regulatory sys-tems also control expression of the pdu and cob op-erons [46]. Aerobic induction by propanediolrequires Crp/cAMP, and is inhibited by glucoseand glycerol. Anaerobic expression is controlled byboth Crp/cAMP and the ArcAB two-component sys-tem, which have additive e¡ects.

4. Reaction mechanism

Glycerol and diol dehydratases belong to the classII of coenzyme B12-containing enzymes. Class I com-promises enzymes such as methylmalonyl-CoA mu-tase catalyzing carbon^carbon rearrangements [47].Recent X-ray crystallographic studies of this proteinrevealed that cobalamin is bound to the enzyme via

the imidazole of a histidine residue coordinating tothe cobalt atom in the lower axial position instead ofthe 5,6-dimethyl-benzimidazole moiety of the coen-zyme (base-o¡ form) [48]. The sequence D-x-H-x-x-G, which contains the coordinating histidine residue,is reported to be conserved in this enzyme and someother cobalamin-dependent enzymes. However, thededuced amino acid sequences of the dehydratasesdo not show the B12-binding motif of the class Ienzymes (see Section 6). This fact indicates thatcoenzyme B12 is bound in a di¡erent manner. EPRmeasurements with 15N-labeled dehydratase apoen-zyme and unlabeled coenzyme suggests that cobala-min is bound to diol dehydratase with the 5,6-di-methylbenzimidazole ligand coordinating to thecobalt atom (base-on form) [4,49].

The reaction mechanism of glycerol and diol de-hydratases can be formulated according to Abelesand coworkers [3,4] as depicted in Fig. 3. Binding

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Fig. 3. Mechanism of glycerol dehydratase reaction (adapted from [4]). The coenzyme B12 is depicted in the base-on form. Ade, adenine;RCH2, adenosyl ; [Co], cobalamin; E, enzyme.


of the coenzyme to the apoenzyme activates the Co-C bond of the coenzyme. The substrate-induced ho-molytic cleavage of the Co-C bond leads to the for-mation of cob(II)alamin and an adenosyl radical.The adenosyl radical plays an essential part in thecatalysis by abstracting a hydrogen atom from thesubstrate. This leads to the formation of a substrate-derived radical and 5P-deoxyadenosine. The sub-strate-derived radical then rearranges to a productradical by a hydroxyl group transfer from C-2 toC-1. The product radical abstracts a hydrogenatom back from 5P-deoxyadenosine. This results inthe formation of the ¢nal product and regenerationof the coenzyme.

The radical intermediates formed during the cata-lytic cycle must sustain their high reactivity at theactive site and must become extinct in the onlyway destined for the reaction. Once a radical inter-mediate is quenched by undesirable side reactions orescapes from the active site, regeneration of thecoenzyme is impossible. This leads not only to ces-sation of the catalytic cycle, but also to inactivationof the enzyme, since the modi¢ed coenzyme remainstightly bound to the enzyme and is not exchangeablewith free intact coenzyme B12. Therefore, the radicalspecies must be strictly protected from side reactionsor from leaving the reaction center of the enzyme.Undesirable side reactions occur in the case of glyc-erol and diol dehydratases and these enzymes aresubject to mechanism-based suicide inactivation byglycerol and some other substrates [2,13,50,51]. In-activation by glycerol involves irreversible cleavageof the Co-C bond of coenzyme B12, forming 5P-de-oxyadenosine and an alkylcobalamin-like species. Ir-reversible inactivation is then brought about by tightbinding of the modi¢ed coenzyme [50].

Such suicide inactivation seems enigmatic, becauseglycerol is a growth substrate and the dehydratase isessential for glycerol breakdown (see Section 3.1).Work of Toraya and co-workers [43,44] showedthat glycerol-inactivated dehydratase undergoes rap-id reactivation in permeabilized cells (in situ) by ex-change of the modi¢ed coenzyme for intact coen-zyme B12 in the presence of ATP and Mg2� orMn2�. Recently, two proteins (DdrA and DdrB),which are probably involved in the reactivation re-action, were identi¢ed in K. oxytoca [44] and oneprotein (OrfZ) in C. freundii [35].

5. Assay systems

For determination of glycerol or diol dehydrataseactivity, the 3-methyl-2-benzothialzolinone hydra-zone (MBTH) method is widely applied [52]. Thismethod is based on the ability of the aldehydesformed during the dehydratase reaction to reactwith MBTH. The resulting azine derivatives are de-tected spectrophotometrically. The usual assay mix-ture contains an appropriate amount of dehydratase,0.2 M substrate (i.e. 1,2-propanediol), 0.05 M KCl,0.035 M potassium phosphate bu¡er (pH 8.0), and15 WM coenzyme B12, in a total volume of 1 ml.After incubation at 37³C for 1^10 min, the enzymereaction is terminated by adding 1 ml 0.1 M potas-sium citrate bu¡er (pH 3.6) and 0.5 ml of MBTHhydrochloride. After 15 min at 37³C, the amount ofaldehyde formed (i.e. propionaldehyde) is deter-mined from the absorbance at 305 nm. The apparentmolar extinction coe¤cient at 305 nm for the coloredproduct from propionaldehyde is 13.3U103 M31

cm31. The MBTH method is suitable for variousapplications, such as kinetic and mechanistic studies,determination of substrate and cofactor speci¢city.Because of its higher brevity and sensitivity, theMBTH method replaced the 2,4-dinitrophenylhydra-zine assay [52], but the latter compound is still ap-plied for the identi¢cation of glycerol or diol dehy-dratase in polyacrylamide gels after separation ofcrude extracts or protein preparations by electropho-resis under non-denaturing conditions [1,11,14,37].The dehydratase band can be localized by the col-ored precipitate of the 2,4-dinitrophenylhydrazone ofpropionaldehyde [11].

Another assay is a coupled reaction in which thepropionaldehyde formed during the glycerol or dioldehydratase reaction with 1,2-propanediol as sub-strate is reduced to 1-propanol in the presence ofadded excess L-NADH and yeast alcohol dehydro-genase. The decrease in the absorbance of NADH at340 nm is used for calculation of the dehydrataseactivity [50]. This method is suitable when 1,2-pro-panediol is used as substrate and the appropriateassay conditions allow alcohol dehydrogenase activ-ity.

A major problem during dehydratase assays is therapid inactivation of the enzyme with glycerol assubstrate. This problem can be circumvented by us-

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Fig. 4. Alignment of the amino acid sequences corresponding to the K, L and Q subunits of glycerol and diol dehydratases from variousorganisms. The dhaBCE genes of Cl. pasteurianum (C. p.) [37] and C. freundii (C. f.) [1,13] and the gldABC genes of K. pneumoniae (K.p.) [14] encode the K, L and Q subunits of coenzyme B12-dependent glycerol dehydratases. The pddABC genes of K. oxytoca (K. o.) [11]and the pduCDE genes of S. typhimurium (S. t.) [23] encode the K, L and Q subunits of coenzyme B12-dependent diol dehydratases.Dashed lines indicate gaps which were introduced to optimize the alignment. The amino acids conserved in all ¢ve enzymes are shaded.


ing 1,2-propanediol as substrate or a short reactiontime (1 min). Work of Toraya and Fukui [16]showed that the use of 1,2-propanediol does not re-sult in signi¢cant enzyme inactivation and that thereaction with glycerol is linear for approximately1 min.

6. Biochemistry and molecular genetics ofB12-dependent dehydratases

The glycerol dehydratases of K. pneumoniae andC. freundii and the diol dehydratase of K. oxytocaare biochemically well studied. Most of the datasummarized in this section are derived from thework with these three enzymes. Glycerol and themutually related diol dehydratases convert glycerol,1,2-propanediol and 1,2-ethanediol to the corre-sponding aldehydes, with glycerol being the preferredsubstrate for glycerol dehydratase and 1,2-propane-diol being the preferred substrate for diol dehydra-tase [2,37,38,53,54]. All characterized dehydratasesconsist of three types of subunits and have the sub-unit composition K2L2Q2 [1,55]. The native enzymecomplex of glycerol dehydratase and diol dehydra-tase dissociates into components A and B or F and Swhen subjected to ion-exchange chromatography[11,56,57]. Recent studies with the diol dehydrataseof K. oxytoca identi¢ed the components F and S as Lsubunit and K2Q2 complex, respectively. The K and Qsubunits require each other for correct folding form-ing the soluble, active component S. Expression ofcomponent F in a soluble, active form is promotedby coexpression with both the K and Q subunits,probably by coexistence with component S [55].

The reported native molecular mass of glycerol de-hydratase (approximately 190 kDa) [1,37,53] is lowerthan that of diol dehydratase (approximately 230kDa) [12,55]. Both types of dehydratases are acti-vated by certain monovalent cations; K�, NH4

�

and Rb� are most e¡ective as cofactor [13,58]. Incontrast to glycerol dehydratase, diol dehydrataseis also partially active with Cs� and Na�. Glyceroland diol dehydratases of various Klebsiella andCitrobacter strains are distinguishable by their di¡er-ent immunochemical properties [16] and Km valuesfor coenzyme B12 (13^50 nM and 750^1400 nM, re-spectively) [13,15,55,59].

The DNA sequence for diol dehydratase has beendetermined in two organisms and that for glyceroldehydratase in three [1,11,13,14,23,37]. Each enzymeis encoded by three genes that are transcribed in thefollowing order: large gene (K subunit); intermediategene (L subunit) ; small gene (Q subunit). The de-duced molecular masses of the subunits are approx-imately 60.5, 19.5^24 and 16^19 kDa, respectively.The intermediate and the small subunits of diol de-hydratases possess a higher molecular mass than thecorresponding subunits of glycerol dehydratases (Ta-ble 1). This is caused by additional amino acids inthe N-terminal region of both diol dehydratase sub-units (Fig. 4). Each subunit has signi¢cant aminoacid sequence homology to the analogous subunitfrom the other organisms (Fig. 4). No signi¢cantsimilarities of the dehydratases to other cobalamin-dependent enzymes or cobalamin-binding proteinsare apparent, and no putative binding motif forcoenzyme B12 matching the conserved sequence de-scribed for other cobalamin-dependent proteins [60]is evident.

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Table 1Properties of the genes and the corresponding gene products for the three structural subunits of glycerol and diol dehydratases

Organism Gene name Gene length Protein molecular mass (Da)

K L Q K L Q K L Q

Clostridium pasteurianum dhaB dhaC dhaE 1665 540 441 60 813 19 549 16 722Citrobacter freundii dhaB dhaC dhaE 1668 585 429 60 433 21 487 16 121Klebsiella pneumoniae gldA gldB gldC 1668 585 426 60 621 21 310 16 094Klebsiella oxytoca pddA pddB pddC 1665 675 522 60 348 24 113 19 173Salmonella typhimurium pduC pduD pduE 1665 675 522 60 307 24 157 19 131

The dhaBCE genes of Cl. pasteurianum [37] and C. freundii [1] and the gldABC genes of K. pneumoniae [14] encode coenzyme B12-dependentglycerol dehydratases. The pddABC genes of K. oxytoca [11] and the pduCDE genes of S. typhimurium [23] encode coenzyme B12-dependentdiol dehydratases. K, large subunit; L, intermediate subunit; Q, small subunit.


In some cases, the DNA sequences that £ank dioland glycerol dehydratase have also been determined.Where the sequence downstream of the dehydratasegenes is published, an open reading frame followswhich encodes a polypeptide of approximately 600amino acids (OrfZ, DhaB4 (Orf4), DdrA or Orf1)[1,13,14,37,44]. The sequence comparison of theorfZ gene product of C. freundii revealed 84, 63and 59% identity to the corresponding homologousgene products of K. pneumoniae, Cl. pasteurianumand K. oxytoca, respectively. Since deletion of orfZhas no e¡ect on enzyme activity, it was concludedthat orfZ does not encode a subunit required forglycerol dehydratase activity [1]. Similar resultswere obtained for the dehydratase of K. pneumoniae,K. oxytoca and Cl. pasteurianum [11,14,37]. How-ever, Northern blot experiments performed with C.freundii RNA revealed a common transcription ofthe orfZ gene and the genes encoding the three struc-tural subunits of glycerol dehydratase [35]. Work ofMori et al. [44] showed that the OrfZ homolog DdrAis involved in the reactivation of glycerol- or cyano-cobalamin-inactivated diol dehydratase of K. oxyto-ca.

Other genes that £ank glycerol dehydratase areinvolved in the pathway of glycerol fermentation(dhaD, dhaK, dhaT), or have unknown functions([1,10,32,37]; see Section 3.1). In both, C. freundiiand Cl. pasteurianum, there are three such putativegenes that are conserved between the two organisms,orfW, orfX, orfY (Fig. 1B,C).

Genes adjacent to the diol dehydratase genes of K.oxytoca and S. typhimurium are conserved. This isupstream pduB in S. typhimurium and orf1 in K. oxy-toca with unknown function [11,23] and downstreamthe already mentioned genes involved in reactivationof diol dehydratase. Conservation of the genetic or-ganization for the remaining genes involved in pro-panediol degradation is uncertain since the DNAsequences are incomplete.

7. Dehydratase in biotechnology

1,3-Propanediol is of industrial importance, be-cause it can be used as a starting material for pro-ducing plastics, such as polyesters, polyethers andpolyurethanes. Therefore, industrial interest emerged

to develop improved routes for 1,3-propanediol pro-duction. A production process for 1,3-propanediolfrom glycerol relies on the activity of glycerol ordiol dehydratases, which convert glycerol to 3-hy-droxypropionaldehyde (see Section 3.1). It has beenshown that the dehydratase activity is the limitingfactor for the biotechnological production of 1,3-propanediol [9,61]. This is most likely related tothe role of coenzyme B12 in the catalytic cycle andthe reaction inactivation with glycerol as substrate.Thus, increasing the level of active dehydratase inmicroorganisms could increase productivity. Toachieve this, the genes for the dehydratase could beoverexpressed in 1,3-propanediol-producing organ-isms or the reactivation reaction of glycerol-inacti-vated dehydratase could be improved. As mentionedabove, it has been shown that the genes downstreamof the three structural genes for dehydratase, e.g.orfZ, are not necessary for enzyme activity, but aresomehow involved in reactivation of the enzyme.Work of Skraly et al. [34] revealed that the 1,3-pro-panediol synthesis is more e¡ective in the presence ofthese genes.

8. Conclusions

Signi¢cant progress has been made in recent yearsto understand the dehydratases acting on glycerol ordiols. This came from the elucidation of the role ofcoenzyme B12 in these reactions and from the iden-ti¢cation of the genes coding for these enzymes.There still are a number of questions to be solvedby future work. The binding motif for coenzyme B12

has to be identi¢ed. This and the elucidation of theprotein structure may aid in understanding whythese enzymes are inactivated by glycerol. Also, theexact function of the reactivating enzymes, e.g. OrfZ,has to be unravelled. Further understanding of thedehydratase reactions might also contribute to thedevelopment of an economically feasible processfor 1,3-propanediol production from glycerol.

Acknowledgments

The work of G. Gottschalk and R. Daniel wassupported by the Deutsche Forschungsgemeinschaft

FEMSRE 622 25-1-99


within the Forschungsschwerpunkt `Neuartige Reak-tionen und Katalysemechanismen bei anaerobenMikroorganismen', by the Fonds der ChemischenIndustrie and by the Akademie der Wissenschaften,Goëttingen, Germany.

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Biochemistry of coenzyme B 12 -dependent glycerol and diol dehydratases and organization of the encoding genes

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