Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Fermentative degradation of triethanolamine by a homoacetogenic bacterium

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9±1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B₁₂) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.Abbreviation NTA nitrilotriacetate     

Fermentation of polyethylene glycol via acetaldehyde in Pelobacter venetianus

Summary Pelobacter venetianus, a strictly anaerobic bacterium recently isolated with polyethylene glycol (PEG) as substrate, ferments PEG's with molecular masses of 106–40000, as well as acetoin, ethanolamine, choline, and ethoxyethanol, to acetate and ethanol. Ethylene glycol (EG) and acetaldehyde were fermented in the same manner at limiting concentrations in continuous culture. Growth with glycolaldehyde led to acetate as sole fermentation product. Acetaldehyde appeared as byproduct of PEG fermentation, and accumulated to high concentrations during degradation of PEG 4000 and PEG 6000. Utilization of PEG's was constitutive, whereas acetoin degradation was inducible. Acetaldehyde was shown to be the primary product of EG degradation, and inhibited utilization of other substrates. Enzymes involved in the fermentation of PEG, EG, acetoin, and glycolaldehyde were demonstrated in cell-free extracts, except for the PEG degrading enzyme and EG dehydrase. These results demonstrate that acetaldehyde plays a central role in the metabolism of Pelobacter venetianus. A scheme of intermediary metabolism and PEG degradation is discussed.Abbreviations EG ethylene glycol - Di-EG diethylene glycol - PEG (20 000) polyethylene glycol (molecular weight 20 000)     

Low-solubility glycerol dehydratase,a chimeric enzyme of coenzyme B<Subscript>12</Subscript>-dependent glycerol and diol dehydratases

Coenzyme B₁₂-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the β and γ subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the β and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the β and γ subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase β subunit in addition to the diol dehydratase-specific extra-regions of β and γ subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short N-terminal sequences are sufficient to change the solubility of the enzyme.     

Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

To isolate genes encoding coenzyme B₁₂-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α₂β₂γ₂) structure with a native molecular mass of 210 kDa. It requires coenzyme B₁₂ for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K _m values for coenzyme B₁₂, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B₁₂-dependent diol dehydratase and not a glycerol dehydratase.     

Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium

A strictly anaerobic gram-positive, rod-shaped bacterium, strain LuPhet1, was isolated from sewage sludge with phenoxyethanol as sole carbon and energy source, and was assigned to the genus Acetobacterium. The new isolate fermented the alkylaryl ether compound phenoxyethanol stoichiometrically to phenol and acetate, whereas phenoxyacetic acid was not degraded. In cell-free extracts of strain LuPhet1, cleavage of the ether linkage was shown, and acetaldehyde was detected as reaction product. Coenzyme A-dependent acetaldehyde: acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results indicate that the ether linkage of phenoxyethanol is cleaved by a shift of the hydroxyl group to the subterminal carbon atom, analogous to a corrinoid-dependent diol dehydratase reaction, to form an unstable hemiacetal that releases phenol and acetaldehyde. Obviously, phenoxyethanol is degraded by the same strategy as in anaerobic degradation of the alkyl ether polyethylene glycol.     

Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase.

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-B12-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-B12-dependent diol dehydratase in a 1,2-propanediol-containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.     

Microbial degradation of polyethers

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.     

The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.     

Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases

Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.     

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.     

Insights into the genome of the enteric bacterium Escherichia blattae: cobalamin (B12) biosynthesis, B12-dependent reactions, and inactivation of the gene region encoding B12-dependent glycerol dehydratase by a new mu-like prophage

The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B12) biosynthesis and B12-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B12 de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B12 biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B12-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B12-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B12-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B12-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B12-dependent enzyme, the methylcobalamin-dependent methionine synthase.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol

2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria (Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations.     

Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases.

Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source.     

Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203

Polyethylene glycol (PEG) 4000-utilizing bacterium no. 203 was identified as a Flavobacterium species. 2, 6-Dichlorophenol-indophenol (DCIP)-dependent PEG dehydrogenase was constitutively formed in nutrient broth, glucose and PEG media. However, the enzyme formation was repressed in the presence of an excess amount (over 0.25%) of PEG 400 or 1000. PEG dehydrogenase was purified approximately 34 fold by precipitation with ammonium sulfate, solubilization with benzalkonium chloride, chromatography with DEAE-Toyopearl 650 M and hydroxylapatite and gel filtration on Toyopearl HW-55. The molecular weight of the purified PEG dehydrogenase was calculated to be approximately 2.20 × 10⁵, a value which seemed to consist of four subunits with the same molecular weight of 5.70 × 10⁴. The enzyme was stable below 40°C and in the pH range of 7.0 and 8.0. The optimum pH and temperature of the activity were around 8.0 and 40°C, respectively. The enzyme reduced DCIP and coenzyme Q₁ and Q₂. PEG dehydrogenase showed activity toward various PEG molecules (dimer-PEG 20,000). The apparent K_m values for PEG 400, 1000, 4000 and 6000 were about 1.0, 1.7, 2.8 and 5.9 mM, respectively. The enzyme oxidized primary aliphatic alcohols of C₃–C₁₂, the corresponding aldehydes of C₃–C₇, aromatic alcohols and aldehydes, diols, etc. The enzyme was inactive on ethylene glycol, glycerol, secondary alcohols and sugar alcohols. The enzyme activity was strongly inhibited by sulfhydryl agents or heavy metals and 1, 4-benzoquinone. The purified enzyme showed absorption apectrum similar to that of PEG 6000 dehydrogenase which has already been reported to be a quinoprotein. The prosthetic group of the enzyme was extracted with methanol and identified as PQQ from its prosthetic group capability for glucose dehydrogenase and the fluorescence spectrum.     

Metabolic Engineering of Klebsiella pneumoniae for the Production of 2-Butanone from Glucose

2-Butanone is an important commodity chemical of wide application in different areas. In this study, Klebsiella pneumoniae was engineered to directly produce 2-butanone from glucose by extending its native 2, 3-butanediol synthesis pathway. To identify the potential enzyme for the efficient conversion of 2, 3-butanediol to 2-butanone, we screened different glycerol dehydratases and diol dehydratases. By introducing the diol dehydratase from Lactobacillus brevis and deleting the ldhA gene encoding lactate dehydrogenase, the engineered K. pneumoniae was able to accumulate 246 mg/L of 2-butanone in shake flask. With further optimization of culture condition, the titer of 2-butanone was increased to 450 mg/L. This study lays the basis for developing an efficient biological process for 2-butanone production.     

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.