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

Rolf Daniel, Thomas A. Bobik, Gerhard Gottschalk
DOI: http://dx.doi.org/10.1111/j.1574-6976.1998.tb00387.x 553-566 First published online: 1 December 1998

Abstract

Glycerol and diol dehydratases exhibit a subunit composition of α2β2γ2 and contain coenzyme B12 in the base-on form. The dehydratase reaction proceeds via a radical mechanism. The dehydratases are subject to reaction inactivation by the substrate glycerol which is caused by a cessation of the catalytic cycle because coenzyme B12 is not regenerated, instead 5′-deoxyadenosine and a catalytically inactive cobalamin are formed. The genetic organization of the dehydratase genes is quite similar in all organisms. Downstream of the dehydratase genes an open reading frame encoding a polypeptide of approximately 600 amino acids was identified which is apparently involved in the reactivation of suicide-inactivated enzyme.

Keywords
  • Glycerol dehydratase
  • Diol dehydratase
  • Coenzyme B12
  • Glycerol utilization
  • 1,2-Propanediol degradation
  • dha regulon
  • pdu operon

1 Introduction

Glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) and diol dehydratase (d,l-1,2-propanediol hydro-lyase, EC 4.2.1.28) can each catalyze the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes [1,2]. These enzymic reactions are known to proceed by a radical mechanism involving coenzyme B12 as an essential cofactor. Coenzyme B12 contains a unique covalent Co-C bond, which is stable in aqueous solution, but easily undergoes homolytic cleavage in coenzyme B12-dependent enzymatic reactions. This homolytic cleavage is the initial step in the catalytic cycle of all coenzyme B12-dependent enzymatic processes [3,4].

The coenzyme B12-dependent glycerol and diol dehydratases are involved in the anaerobic utilization of small molecules, they catalyze molecular rearrangements that generate an aldehyde which can be reduced (i.e. glycerol fermentation) or dismutated to more oxidized and more reduced compounds (i.e. 1,2-propanediol fermentation). Coenzyme B12-containing ethanolamine ammonia-lyase of enteric bacteria converts ethanolamine to acetaldehyde and ammonia, the aldehyde is proposed to dismutate to acetate and ethanol, reactions allowing the generation of ATP in the acetate kinase reaction [5,6]. Analogous reactions take place following the conversion of 1,2-propanediol to propionaldehyde by diol dehydratase [68]. The anaerobic degradation of glycerol is initiated by two enzymes, glycerol dehydrogenase forms dihydroxyacetone and glycerol dehydratase yields 3-hydroxypropionaldehyde which is converted further to 1,3-propanediol [1,9,10]. Glycerol dehydratases and the closely related diol dehydratases have been extensively studied in genera of Enterobacteriaceae such as Klebsiella and Citrobacter. It has been shown that these enzymes are similar in molecular masses and substrate spectra, but are different in monovalent cation selectivity patterns, affinity for coenzyme B12, and substrate specificity [1, 1116].

2 Distribution of glycerol and diol dehydratases

The presence of glycerol and/or diol dehydratases was shown in several Gram-positive and Gram-negative microorganisms, including enteric and propionic acid bacteria, solventogenic clostridia and lactobacilli [7, 15, 1727]. All characterized dehydratases are coenzyme B12-dependent, except the diol dehydratase of Clostridium glycolicum[28]. This enzyme seems to be entirely different from the B12-containing dehydratases and will not be discussed here any further. In the case of enteric bacteria, B12-dependent glycerol or diol dehydratases were detected in some strains of the genera Salmonella, Klebsiella, Enterobacter and Citrobacter[7,15,18,22,23,25,29]. Both dehydratases may occur individually or together in these organisms. The 1,2-propanediol- and non-glycerol-fermenting Salmonella typhimurium contain diol dehydratase exclusively. The glycerol-fermenting Enterobacter agglomerans, Klebsiella pneumoniae and Citrobacter freundii possess glycerol dehydratase, some strains of the latter two microorganisms also possess diol dehydratase. Growth on 1,2-propanediol induces only the diol dehydratase, whereas both dehydratases are present in anaerobically grown glycerol cells [15]. In one Klebsiella strain (ATCC 8724), the isofunctional diol dehydratase substitutes for the defective glycerol dehydratase [2,16,22].

Glycerol dehydratase was detected in Clostridium pasteurianum, Cl. butyricum, Lactobacillus sp., Lactobacillus reuteri, Lactobacillus brevis and Lactobacillus buchneri[19,20,24,27,30] and diol dehydratase in the propionic acid bacterium Propionibacterium freudenreichii[22].

3 Physiology and regulation of dehydratase production

3.1 Glycerol fermentation

The pathway of glycerol breakdown and the key enzymes and genes involved have been extensively studied in C. freundii and K. pneumoniae. Glycerol is converted by these bacteria to 1,3-propanediol (major product), ethanol, 2,3-butanediol, acetic and lactic acids [31]. In the absence of an external oxidant, glycerol is fermented by a dismutation process involving two pathways, one serving for glycerol oxidation, the other for the consumption of reducing equivalents generated (Fig. 1A). Oxidation of glycerol is catalyzed by NAD+-linked glycerol dehydrogenase which converts the substrate to dihydroxyacetone. This product is then phosphorylated by dihydroxyacetone kinase and funneled into the glycolytic pathway [10]. Generation of NAD+ is achieved by the NADH-linked 1,3-propanediol dehydrogenase [32]. By the action of coenzyme B12-dependent glycerol dehydratase glycerol is first converted to 3-hydroxypropionaldehyde [1] which then is reduced to 1,3-propanediol accounting for about 50–66% of the glycerol consumed. All four key enzymes and the corresponding genes have been identified and characterized in C. freundii and K. pneumoniae[1, 10, 18, 21, 3234]. In C. freundii, the structural genes of the glycerol dehydratase (dhaBCE) are part of the dha regulon along with the genes encoding the three other key enzymes (dhaD, dhaK, dhaT) of the pathway (Fig. 1B). In addition, the dha regulon encodes a transcriptional activator protein (DhaR) [10], a protein probably involved in reactivation of glycerol dehydratase (OrfZ) [35], and three presumptive proteins with unknown function (OrfW, OrfX, OrfY). The expression of the dha regulon is induced under anaerobic conditions when dihydroxyacetone or glycerol are present. In contrast to the 1,3-propanediol-forming enteric bacteria, very little information is available about the genes and enzymes responsible for glycerol utilization by clostridia and lactobacilli.

1

Anaerobic utilization of glycerol. (A) Pathway used by enteric bacteria. (B) Apparent genetic organization of the C. freundii dha regulon. (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

The glycerol fermentation pattern of clostridia is different, they form butyric acid and some strains of Cl. pasteurianum butanol and ethanol as additional products ([17,30,36], for the entire pathway of solvent formation see [17]). All four key enzymes known from the study of enteric bacteria were detected in crude extracts of Cl. pasteurianum[19] as well as in Cl. butyricum, except that the dihydroxyacetone kinase was not measured in the latter organism [20]. These results indicate that clostridia ferment glycerol like the 1,3-propanediol-forming enteric bacteria by a dismutation process. Recently, the genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase of Cl. pasteurianum were identified [19,37]. The deduced amino acid sequences and the properties of the gene products are very similar to those of 1,3-propanediol-forming enteric bacteria, but the genetic organization is different (Fig. 1B,C). In contrast to C. freundii and K. pneumoniae (not shown) the genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase of Cl. pasteurianum showed the same orientation and all presumptive genes were located upstream of the dhaT gene.

The participation of glycerol dehydratase and 1,3-propanediol dehydrogenase in glycerol fermentation has also been shown for some Lactobacillus species such as L. reuteri, L. buchneri and L. brevis[24,26,27]. In contrast to the mentioned enteric bacteria and clostridia, lactobacilli are not able to grow on glycerol as sole carbon and energy source. These organisms require a second substrate such as glucose for glycerol utilization because of the absence of the enzymes for the oxidative branch.

3.2 1,2-Propanediol fermentation

Diol dehydratase is used by some enteric and propionic acid bacteria for the degradation of 1,2-propanediol [7,22]. Catabolism of this small molecule is likely to be important in a variety of nutritionally complex environments. 1,2-Propanediol is an end-product of the fermentation of rhamnose and fucose. These sugars are common in plant cell walls and are also found in the glycoconjugates of mammalian intestinal epithelial cells. Diol dehydratase catalyzes the first step in the pathway of propanediol degradation, which produces propionaldehyde [38]. Subsequently, propionaldehyde is converted to equal amounts of propanol and propionic acid. Coenzyme A-dependent aldehyde dehydrogenase, phosphotransacylase, propionate kinase, and alcohol dehydrogenase are proposed to catalyze this disproportionation (Fig. 2A) [7,8]. Fermentation of 1,2-propanediol provides one ATP per molecule of propanediol, but no source of carbon. Aerobically, 1,2-propanediol can provide both carbon and energy. It is thought that the aerobic and anaerobic pathways are similar, but that oxygen allows the conversion of some propionyl-CoA to cell carbon.

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. The pduGHJ genes have not yet been correlated to the physical map. ORFs 1–15 include homologs of the diol dehydratase reactivating protein (DdrA) of K. oxytoca, alcohol dehydrogenase, acetate kinase, as well as four homologs of carboxysome shell protein genes.

In S. enterica (formerly called S. typhimurium) the structural genes for diol dehydratase (pduCDE) are located in the pdu operon along with at least 20 additional genes involved in propanediol degradation (Fig. 2B) [39,40]. The pdu operon encodes a propanediol diffusion facilitator (PduF), a transcriptional activator protein (PocR), and homologs of acetate kinase and alcohol dehydrogenase [3941]. In addition, the pdu operon includes genes for the reactivation of diol dehydratase, genes for the conversion of cobalamins to coenzyme B12, and genes that are homologs of the shell proteins of carboxysomes (polyhedral bodies that encase Rubisco and which are thought to function in concentrating CO2) [4042].

The apparent use of the carboxysome shell protein homologs is to encase diol dehydratase within a polyhedral shell. Thus far, four homologs of carboxysome shell protein genes have been identified in the pdu operon [40]. Electron microscopy showed that S. enterica forms polyhedral bodies during growth on 1,2-propanediol, and immuno-electron microscopy indicated that diol dehydratase was located within these polyhedra. The physiological reason for encasement of diol dehydratase within these polyhedral shells is currently unknown. S. enterica is not an autotroph, it does not express Rubisco, and there is no known role for CO2 in the degradation of 1,2-propanediol.

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

In S. enterica, expression of diol dehydratase is regulated via transcriptional control of the pdu operon. Both 1,2-propanediol and poor growth conditions are required for high expression. In addition, induction of the pdu operon is coordinated with induction of adjacent cobalamin biosynthesis (cob) operon [45]. Co-expression of the pdu and cob operons is achieved through the PocR, an activator protein which mediates induction of both operons in response to propanediol. Two global regulatory systems also control expression of the pdu and cob operons [46]. Aerobic induction by propanediol requires Crp/cAMP, and is inhibited by glucose and glycerol. Anaerobic expression is controlled by both Crp/cAMP and the ArcAB two-component system, which have additive effects.

4 Reaction mechanism

Glycerol and diol dehydratases belong to the class II of coenzyme B12-containing enzymes. Class I compromises enzymes such as methylmalonyl-CoA mutase catalyzing carbon–carbon rearrangements [47]. Recent X-ray crystallographic studies of this protein revealed that cobalamin is bound to the enzyme via the imidazole of a histidine residue coordinating to the cobalt atom in the lower axial position instead of the 5,6-dimethyl-benzimidazole moiety of the coenzyme (base-off 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 some other cobalamin-dependent enzymes. However, the deduced amino acid sequences of the dehydratases do not show the B12-binding motif of the class I enzymes (see Section 6). This fact indicates that coenzyme B12 is bound in a different manner. EPR measurements with 15N-labeled dehydratase apoenzyme and unlabeled coenzyme suggests that cobalamin is bound to diol dehydratase with the 5,6-dimethylbenzimidazole ligand coordinating to the cobalt atom (base-on form) [4,49].

The reaction mechanism of glycerol and diol dehydratases can be formulated according to Abeles and coworkers [3,4] as depicted in Fig. 3. Binding of the coenzyme to the apoenzyme activates the Co-C bond of the coenzyme. The substrate-induced homolytic cleavage of the Co-C bond leads to the formation of cob(II)alamin and an adenosyl radical. The adenosyl radical plays an essential part in the catalysis by abstracting a hydrogen atom from the substrate. This leads to the formation of a substrate-derived radical and 5′-deoxyadenosine. The substrate-derived radical then rearranges to a product radical by a hydroxyl group transfer from C-2 to C-1. The product radical abstracts a hydrogen atom back from 5′-deoxyadenosine. This results in the formation of the final product and regeneration of the coenzyme.

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.

The radical intermediates formed during the catalytic cycle must sustain their high reactivity at the active site and must become extinct in the only way destined for the reaction. Once a radical intermediate is quenched by undesirable side reactions or escapes from the active site, regeneration of the coenzyme is impossible. This leads not only to cessation of the catalytic cycle, but also to inactivation of the enzyme, since the modified coenzyme remains tightly bound to the enzyme and is not exchangeable with free intact coenzyme B12. Therefore, the radical species must be strictly protected from side reactions or from leaving the reaction center of the enzyme. Undesirable side reactions occur in the case of glycerol and diol dehydratases and these enzymes are subject to mechanism-based suicide inactivation by glycerol and some other substrates [2,13,50,51]. Inactivation by glycerol involves irreversible cleavage of the Co-C bond of coenzyme B12, forming 5′-deoxyadenosine and an alkylcobalamin-like species. Irreversible inactivation is then brought about by tight binding of the modified coenzyme [50].

Such suicide inactivation seems enigmatic, because glycerol is a growth substrate and the dehydratase is essential for glycerol breakdown (see Section 3.1). Work of Toraya and co-workers [43,44] showed that glycerol-inactivated dehydratase undergoes rapid reactivation in permeabilized cells (in situ) by exchange of the modified coenzyme for intact coenzyme B12 in the presence of ATP and Mg2+ or Mn2+. Recently, two proteins (DdrA and DdrB), which are probably involved in the reactivation reaction, were identified in K. oxytoca[44] and one protein (OrfZ) in C. freundii[35].

5 Assay systems

For determination of glycerol or diol dehydratase activity, the 3-methyl-2-benzothialzolinone hydrazone (MBTH) method is widely applied [52]. This method is based on the ability of the aldehydes formed during the dehydratase reaction to react with MBTH. The resulting azine derivatives are detected spectrophotometrically. The usual assay mixture contains an appropriate amount of dehydratase, 0.2 M substrate (i.e. 1,2-propanediol), 0.05 M KCl, 0.035 M potassium phosphate buffer (pH 8.0), and 15 μM coenzyme B12, in a total volume of 1 ml. After incubation at 37°C for 1–10 min, the enzyme reaction is terminated by adding 1 ml 0.1 M potassium citrate buffer (pH 3.6) and 0.5 ml of MBTH hydrochloride. After 15 min at 37°C, the amount of aldehyde formed (i.e. propionaldehyde) is determined from the absorbance at 305 nm. The apparent molar extinction coefficient at 305 nm for the colored product from propionaldehyde is 13.3×103 M−1 cm−1. The MBTH method is suitable for various applications, such as kinetic and mechanistic studies, determination of substrate and cofactor specificity. Because of its higher brevity and sensitivity, the MBTH method replaced the 2,4-dinitrophenylhydrazine assay [52], but the latter compound is still applied for the identification of glycerol or diol dehydratase in polyacrylamide gels after separation of crude extracts or protein preparations by electrophoresis under non-denaturing conditions [1,11,14,37]. The dehydratase band can be localized by the colored precipitate of the 2,4-dinitrophenylhydrazone of propionaldehyde [11].

Another assay is a coupled reaction in which the propionaldehyde formed during the glycerol or diol dehydratase reaction with 1,2-propanediol as substrate is reduced to 1-propanol in the presence of added excess β-NADH and yeast alcohol dehydrogenase. The decrease in the absorbance of NADH at 340 nm is used for calculation of the dehydratase activity [50]. This method is suitable when 1,2-propanediol is used as substrate and the appropriate assay conditions allow alcohol dehydrogenase activity.

A major problem during dehydratase assays is the rapid inactivation of the enzyme with glycerol as substrate. This problem can be circumvented by using 1,2-propanediol as substrate or a short reaction time (1 min). Work of Toraya and Fukui [16] showed that the use of 1,2-propanediol does not result in significant enzyme inactivation and that the reaction with glycerol is linear for approximately 1 min.

6 Biochemistry and molecular genetics of B12-dependent dehydratases

The glycerol dehydratases of K. pneumoniae and C. freundii and the diol dehydratase of K. oxytoca are biochemically well studied. Most of the data summarized in this section are derived from the work with these three enzymes. Glycerol and the mutually related diol dehydratases convert glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes, with glycerol being the preferred substrate for glycerol dehydratase and 1,2-propanediol being the preferred substrate for diol dehydratase [2,37,38,53,54]. All characterized dehydratases consist of three types of subunits and have the subunit composition α2β2γ2[1,55]. The native enzyme complex of glycerol dehydratase and diol dehydratase dissociates into components A and B or F and S when subjected to ion-exchange chromatography [11,56,57]. Recent studies with the diol dehydratase of K. oxytoca identified the components F and S as β subunit and α2γ2 complex, respectively. The α and γ subunits require each other for correct folding forming the soluble, active component S. Expression of component F in a soluble, active form is promoted by coexpression with both the α and γ subunits, probably by coexistence with component S [55]. The reported native molecular mass of glycerol dehydratase (approximately 190 kDa) [1,37,53] is lower than that of diol dehydratase (approximately 230 kDa) [12,55]. Both types of dehydratases are activated by certain monovalent cations; K+, NH4+ and Rb+ are most effective as cofactor [13,58]. In contrast to glycerol dehydratase, diol dehydratase is also partially active with Cs+ and Na+. Glycerol and diol dehydratases of various Klebsiella and Citrobacter strains are distinguishable by their different immunochemical properties [16] and Km values for coenzyme B12 (13–50 nM and 750–1400 nM, respectively) [13,15,55,59].

The DNA sequence for diol dehydratase has been determined in two organisms and that for glycerol dehydratase in three [1,11,13,14,23,37]. Each enzyme is encoded by three genes that are transcribed in the following order: large gene (α subunit); intermediate gene (β subunit); small gene (γ subunit). The deduced molecular masses of the subunits are approximately 60.5, 19.5–24 and 16–19 kDa, respectively. The intermediate and the small subunits of diol dehydratases possess a higher molecular mass than the corresponding subunits of glycerol dehydratases (Table 1). This is caused by additional amino acids in the N-terminal region of both diol dehydratase subunits (Fig. 4). Each subunit has significant amino acid sequence homology to the analogous subunit from the other organisms (Fig. 4). No significant similarities of the dehydratases to other cobalamin-dependent enzymes or cobalamin-binding proteins are apparent, and no putative binding motif for coenzyme B12 matching the conserved sequence described for other cobalamin-dependent proteins [60] is evident.

View this table:
1

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

4

Alignment of the amino acid sequences corresponding to the α, β and γ subunits of glycerol and diol dehydratases from various organisms. 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 α, β and γ 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 α, β and γ subunits of coenzyme B12-dependent diol dehydratases. Dashed lines indicate gaps which were introduced to optimize the alignment. The amino acids conserved in all five enzymes are shaded.

In some cases, the DNA sequences that flank diol and glycerol dehydratase have also been determined. Where the sequence downstream of the dehydratase genes is published, an open reading frame follows which encodes a polypeptide of approximately 600 amino acids (OrfZ, DhaB4 (Orf4), DdrA or Orf1) [1,13,14,37,44]. The sequence comparison of the orfZ gene product of C. freundii revealed 84, 63 and 59% identity to the corresponding homologous gene products of K. pneumoniae, Cl. pasteurianum and K. oxytoca, respectively. Since deletion of orfZ has no effect on enzyme activity, it was concluded that orfZ does not encode a subunit required for glycerol dehydratase activity [1]. Similar results were obtained for the dehydratase of K. pneumoniae, K. oxytoca and Cl. pasteurianum[11,14,37]. However, Northern blot experiments performed with C. freundii RNA revealed a common transcription of the orfZ gene and the genes encoding the three structural subunits of glycerol dehydratase [35]. Work of Mori et al. [44] showed that the OrfZ homolog DdrA is involved in the reactivation of glycerol- or cyanocobalamin-inactivated diol dehydratase of K. oxytoca.

Other genes that flank glycerol dehydratase are involved in the pathway of glycerol fermentation (dhaD, dhaK, dhaT), or have unknown functions ([1,10,32,37]; see Section 3.1). In both, C. freundii and Cl. pasteurianum, there are three such putative genes 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 is upstream pduB in S. typhimurium and orf1 in K. oxytoca with unknown function [11,23] and downstream the already mentioned genes involved in reactivation of diol dehydratase. Conservation of the genetic organization for the remaining genes involved in propanediol degradation is uncertain since the DNA sequences are incomplete.

7 Dehydratase in biotechnology

1,3-Propanediol is of industrial importance, because it can be used as a starting material for producing plastics, such as polyesters, polyethers and polyurethanes. Therefore, industrial interest emerged to develop improved routes for 1,3-propanediol production. A production process for 1,3-propanediol from glycerol relies on the activity of glycerol or diol dehydratases, which convert glycerol to 3-hydroxypropionaldehyde (see Section 3.1). It has been shown that the dehydratase activity is the limiting factor for the biotechnological production of 1,3-propanediol [9,61]. This is most likely related to the role of coenzyme B12 in the catalytic cycle and the reaction inactivation with glycerol as substrate. Thus, increasing the level of active dehydratase in microorganisms could increase productivity. To achieve this, the genes for the dehydratase could be overexpressed in 1,3-propanediol-producing organisms or the reactivation reaction of glycerol-inactivated dehydratase could be improved. As mentioned above, it has been shown that the genes downstream of the three structural genes for dehydratase, e.g. orfZ, are not necessary for enzyme activity, but are somehow involved in reactivation of the enzyme. Work of Skraly et al. [34] revealed that the 1,3-propanediol synthesis is more effective in the presence of these genes.

8 Conclusions

Significant progress has been made in recent years to understand the dehydratases acting on glycerol or diols. This came from the elucidation of the role of coenzyme B12 in these reactions and from the identification of the genes coding for these enzymes. There still are a number of questions to be solved by future work. The binding motif for coenzyme B12 has to be identified. This and the elucidation of the protein structure may aid in understanding why these enzymes are inactivated by glycerol. Also, the exact function of the reactivating enzymes, e.g. OrfZ, has to be unravelled. Further understanding of the dehydratase reactions might also contribute to the development of an economically feasible process for 1,3-propanediol production from glycerol.

Acknowledgements

The work of G. Gottschalk and R. Daniel was supported by the Deutsche Forschungsgemeinschaft within the Forschungsschwerpunkt ‘Neuartige Reaktionen und Katalysemechanismen bei anaeroben Mikroorganismen’, by the Fonds der Chemischen Industrie and by the Akademie der Wissenschaften, Göttingen, Germany.

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View Abstract