Diol dehydratase: N-terminal amino acid sequences and subunit stoichiometry

N-terminal sequence analysis of diol dehydratase and its constituent subunits shows that the ratio of the 60K:51K:29K:15K subunits in the native enzyme is 2:1:2:2. From the amino acid compositions of the individual subunits diol dehydratase appears to be a peripheral membrane protein.     

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.     

Release of a damaged cofactor from a coenzyme B12-dependent enzyme: X-ray structures of diol dehydratase-reactivating factor

The crystal structures of ADP bound and nucleotide-free forms of molecular chaperone-like diol dehydratase-reactivating factor (DDR) were determined at 2.0 and 3.0 A, respectively. DDR exists as a dimer of heterodimer (alphabeta)2. The alpha subunit has four domains: ATPase domain, swiveling domain, linker domain, and insert domain. The beta subunit, composed of a single domain, has a similar fold to the beta subunit of diol dehydratase (DD). The binding of an ADP molecule to the nucleotide binding site of DDR causes a marked conformational change of the ATPase domain of the alpha subunit, which would weaken the interactions between the DDR alpha and beta subunits and make the displacement of the DDR beta subunit by DD through the beta subunit possible. The binding of the DD beta subunit to the DDR alpha subunit induces steric repulsion between the DDR alpha and DD alpha subunits that would lead to the release of a damaged cofactor from inactivated holoDD.     

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.     

Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor.

The mechanism of reactivation of diol dehydratase by its reactivating factor was investigated in vitro by using enzyme. cyanocobalamin complex as a model for inactivated holoenzyme. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins through intermediate formation of apoenzyme. The factor showed extremely low but distinct ATP-hydrolyzing activity. It formed a tight complex with apoenzyme in the presence of ADP but not at all in the presence of ATP. Incubation of the enzyme.cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin, leaving the tight apoenzyme-reactivating factor complex. Although the resulting complex was inactive even in the presence of added adenosylcobalamin, it dissociated by incubation with ATP, forming the apoenzyme, which was reconstitutable into active holoenzyme with added coenzyme. Thus, it was established that the reactivation of the inactivated holoenzyme by the factor in the presence of ATP and Mg2+ takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme.factor complex. ATP plays dual roles as a precursor of ADP in the first step and as an effector to change the factor into the low-affinity form for diol dehydratase. The enzyme-bound adenosylcobalamin was also susceptible to exchange with free adeninylpentylcobalamin, although to a much lesser degree. The mechanism for discrimination of adenine-containing cobalamins from adenine-lacking cobalamins was explained in terms of formation equilibrium constants of the cobalamin.enzyme.reactivating factor ternary complexes. We propose that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.     

Domain swapping between a cyanobacterial and a plant subunit ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.     

The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment

Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.     

Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by approximately 100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of approximately 1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.     

Movement of the iron-sulfur subunit beyond the ef loop of cytochrome b is required for multiple turnovers of the bc1 complex but not for single turnover Qo site catalysis.

Recent kinetics experiments using mutants of the bc(1) complex (ubihydroquinone-cytochrome c oxidoreductase) iron-sulfur subunit with modified hinge regions have revealed the crucial role played by the large scale movement of its [2Fe-2S] cluster domain during the activity of this enzyme. In particular, one of these mutants (+1Ala) with an insertion of one alanine residue in the hinge region is partially deficient in performing this movement. We found that this defect can be overcome by the appearance of a second mutation substituting the leucine at position 286 in the ef loop of cytochrome b with a phenylalanine. Detailed studies of these mutants and their derivatives revealed that the ef loop acts as a barrier that needs to be crossed for multiple turnovers of the enzyme but not for a single turnover ubihydroquinone oxidation site catalysis. These findings indicate that the movement of the iron-sulfur subunit is composed of two discrete parts: a "micro-movement" at the cytochrome b interface, during which the [2Fe-2S] cluster interacts with ubihydroquinone oxidation site occupants and catalyzes ubihydroquinone oxidation, and a "macro-movement," during which the cluster domain swings away from cytochrome b interface, crosses the ef loop, and reaches a position close to cytochrome c(1) heme, to which it ultimately transfers an electron.     

Interleukin 18 gene polymorphism is a risk factor for multiple sclerosis

Proinflammatory cytokines with immunosuppressive properties play an important role in the pathogenesis of multiple sclerosis (MS). Interleukin 18 (IL-18) is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. The purpose of this study was to determine whether there was any relationship between IL18 gene polymorphisms and MS. IL18 genotyping were performed in 101 MS patients and 164 control subjects by using the PCR–restriction fragment length polymorphism (PCR–RFLP) method. The frequency of MS patients with the CC genotype of the IL18 gene at position ?137 was significantly higher than with the GG genotype [p = 0.01, odds ratio (OR) 3.17]. In haplotype analysis of two SNPs in the IL18 gene, frequency of the CC haplotype was significantly higher in MS patients (p = 0.002, OR 3.0). However, the genotype distribution of the IL18 ?607 C/A polymorphism in the MS patient group was not significantly different from that of the control group. These data suggest that IL18 gene polymorphisms at position ?137 might be a genetic risk factor for MS in the Turkish population.     

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)     

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.     

Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase

Adenosylcobalamin-dependent glycerol dehydratase undergoes mechanism-based inactivation by its physiological substrate glycerol. We identified two genes (gdrAB) of Klebsiella pneumoniae for a glycerol dehydratase-reactivating factor (Tobimatsu, T., Kajiura, H., Yunoki, M., Azuma, M., and Toraya, T. (1999) J. Bacteriol. 181, 4110-4113). Recombinant GdrA and GdrB proteins formed a tight complex of (GdrA)(2)(GdrB)(2), which is a putative reactivating factor. The purified factor reactivated the glycerol-inactivated and O(2)-inactivated glycerol dehydratases as well as activated the enzyme-cyanocobalamin complex in vitro in the presence of ATP, Mg(2+), and adenosylcobalamin. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins in the presence of ATP and Mg(2+) through intermediate formation of apoenzyme. The factor showed extremely low ATP-hydrolyzing activity and formed a tight complex with apoenzyme in the presence of ADP. Incubation of the enzyme-cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin. The resulting tight inactive complex of apoenzyme with the factor dissociated upon incubation with ATP, forming functional apoenzyme and a low affinity form of factor. Thus, it was established that the reactivation of the inactivated holoenzymes takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme-factor complex. We propose that the glycerol dehydratase-reactivating factor is a molecular chaperone that participates in reactivation of the inactivated enzymes.     

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.     

The N-terminal regions of beta and gamma subunits lower the solubility of adenosylcobalamin-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer (alphabetagamma)(2), and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the beta and gamma subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the beta and gamma subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the beta and gamma subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both beta and gamma subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.     

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase

[Omega-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5', which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase were investigated. The kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes.     

Eukaryotic translation initiation factor 3 subunit b is a novel oncogenic factor in prostate cancer

Mammalian Genome - Prostate cancer, the second most common cancer among male adults, affects millions globally. We sought to investigate the expression and contribution of Eukaryotic translation...     

Stereospecificity and mechanism of adenosylcobalamin-dependent diol dehydratase. Catalysis and inactivation with meso- and dl-2,3-butanediols as substrates.

A comparative study of the reaction of meso-, d-, and dl-2,3-butanediols with adenosylcobalamin-dependent dioldehydratase was carried out. While the meso isomer is both a substrate and inactivator of holoenzyme, the d and dl compounds act as purely competitive inhibitors, neither undergoing catalysis nor inactivating holoenzyme. Furthermore, d- and dl-2,3-butanediols protect holoenzyme from oxygen inactivation and enzyme-bound cofactor from photolysis, and do not induce detectable cleavage of the carbon-cobalt bond of cofactor. These results show that the stereospecificity of the inactivation reaction is the same as that of catalysis, suggest that hydrogen abstraction from C-1 of substrate may be concerted with cleavage of the carbon-cobalt bond of adenosylcobalamin, and further suggest that formation of a carbon-cobalt bond between coenzyme and substrate is not obligatory for catalysis.     

Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.