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.     

甘油脱水酶再激活因子提高重组大肠杆菌3-羟基丙酸合成能力

甘油脱水酶是甘油转化3-羟基丙酸生物合成途径中的关键性限速酶,然而底物甘油的存在会抑制该酶的活性,从而引起3-羟基丙酸合成量的下降.因此解除底物甘油对甘油脱水酶活性的抑制作用,是提高生物合成3-羟基丙酸产量的方法之一.克隆来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶编码基因dhaB、甘油脱...     

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.     

Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii.

The coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis. We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase. Northern blot analyses revealed that both genes were expressed during glycerol fermentation. The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3-propanediol dehydrogenase. The dhaF and dhaG gene products were copurified to homogeneity from cell-free extracts of a recombinant E. coli strain producing both His6-tagged proteins. Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da. The subunit structure of the native complex is probably alpha2beta2. The factor rapidly reactivated glycerol- or O2-inactivated hologlycerol dehydratase and activated the enzyme-cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+. The DhaF-DhaG complex and DhaF exhibited ATP-hydrolyzing activity, which was not directly linked to the reactivation of dehydratase. The purified DhaF-DhaG complex of C. freundii efficiently cross-activated the enzyme-cyanocobalamin complex and the glycerol-inactivated glycerol dehydratase of Klebsiella pneumoniae. It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.     

In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase.

The catalytic properties of coenzyme B12-dependent glycerol dehydratase and diol dehydratase were studied in situ with Klebsiella pneumoniae cells permeabilized by toluene treatment, since the in situ enzymes approximate the in vivo conditions of the enzymes more closely than enzymes in cell-free extracts or cell homogenates. Both dehydratases in situ underwent rapid "suicidal" inactivation by glycerol during catalysis, as they do in vitro. The inactivated dehydratases in situ, however, were rapidly and continually reactivated by adenosine 5'-triphosphate (ATP) and Mn2+ in the presence of free adenosylcobalamin, although in cell-free extracts or in cell homogenates they could not be reactivated at all under the same reaction conditions. ATP was partially replaced by cytidine 5'-triphosphate or guanosine 5'-triphosphate but not by the beta, gamma-methylene analog of ATP in the in situ reactivation. Mn2+ was fully replaced by Mg2+ but only partially by Co2+. Hydroxocoblamin could not replace adenosylcobalamin in reactivation mixtures. The ability to reactivate the glycerol-inactivated dehydratases in situ was only seen in cells grown anaerobically in glycerol-containing media. This suggests that some factor(s) required for in situ reactivation is subject to induction by glycerol. Of the two possible mechanisms of in situ reactivation, i.e., the regeneration of adenosylcobalamin by Co-adenosylation of the bound inactivated coenzyme moiety (B12-adenosylation mechanism) and the displacement of the bound inactivated coenzyme moiety by free adenosyl-cobalamin (B12-exchange mechanism), the former seems very unlikely from the experimental results.     

Mechanism of action of adenosylcobalamin: hydrogen transfer in the inactivation of diol dehydratase by glycerol

We have investigated the kinetic characteristics of the inactivation of the adenosylcobalamin-dependent enzyme propanediol dehydratase by glycerol, (RS)-1,1-dideuterioglycerol, (R)-1,1-dideuterioglycerol, and perdeuterioglycerol in the presence of 1,2-propanediol and 1,1-dideuterio-1,2-propanediol. The results imply that hydrogen (or deuterium) attached to C-1 of 1,2-propanediol participates in the inactivation process and contributes to the expression of a kinetic isotope effect on the rate of inactivation. The mechanism for this inactivation must involve the cofactor as an intermediate hydrogen carrier, presumably in the form of 5'-deoxyadenosine. Moreover, a mechanism involving a rate-determining transfer of hydrogen from an intermediate containing three equivalent hydrogens quantitatively accounts for all of the results. When diol dehydratase holoenzyme is inactivated by [1-3H]glycerol, 5'-deoxyadenosine which is enriched in tritium by a factor of 2.1 over that in glycerol can be isolated from the reaction mixture.     

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.     

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.     

Quantitative analysis on inactivation and reactivation of recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li

The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H₁₃, S₁₉₃, N₃₅₉, E₄₀₇, and M₅₁₅ of α subunit, N₄₇, L₁₅₀, V₁₈₉ of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg^?1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O₂ could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg²⁺ and coenzyme B₁₂ were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O₂-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.     

Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous.     

弗氏柠檬酸菌甘油脱水酶激活因子基因的克隆、表达与功能鉴定

1,3-丙二醇是一种重要的化工原料,其生物法生产的研究逐渐受到的关注。研究以弗氏柠檬酸菌的总DNA为模板,通过PCR分别扩增出约1.8kb（dhaF）和0.4kb（dhaG）的两个基因片段分别编码甘油脱水酶激活因子大、小亚基, 连接于pMD-18T载体,测序分析显示与GenBank中相关基因的相似性最高为86%。将两基因以多顺反子的方式与pSE380连接构建表达载体,并在大肠杆菌中进行高效表达,表达量占总蛋白的30%。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化,得到电泳纯级的甘油脱水酶激活因子,SDS-PAGE分析显示：大、小亚基分子量约为63kDa和12kDa;非变性胶分析显示：全酶的分子量约为150kDa,经扫描分析推测甘油脱水酶激活因子很有可能是以α2β2方式结合的。以弗氏柠檬酸菌甘油脱水酶为研究对象,进行激活实验,结果证实该激活因子具备甘油脱水酶激活因子的功能,为进一步阐明甘油脱水酶的激活机制及1,3-丙二醇的高效生产奠定了基础。     

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.     

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.     

An activity stain for dihydroxy-acid dehydratase

An activity stain has been devised for the dihydroxy-acid dehydratase. When applied to polyacrylamide gel electropherograms of crude soluble extracts of Escherichia coli, it detected a single electromorph. The intensity of staining increased with the amount of extract protein applied to the gel. Activity staining demonstrated that (a) anaerobically grown cells contain more extractable dehydratase activity than do aerobically grown cells; (b) exposure of E. coli to 4.2 atm O2 caused virtually complete loss of activity; (c) exposure of cells to paraquat or plumbagin in the presence of dioxygen, but not in its absence, caused a massive loss of activity. These data illustrate the utility of this activity stain and demonstrate that the dehydratase is inactivated by O2- generated within cells.     

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 B(12)-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.     

Resolution of the coenzyme B-12-dependent dehydratases of Klebsiella sp. and Citrobacter freundii.

Diol dehydratase (1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) are shown to be distinct, separable enzymes that occur individually or together in different strains of Klebsiella sp. Anaerobic growth with propan-1,2-diol induces diol dehydratase alone, whereas glycerol fermentation induces both enzymes in K. pneumoniae ATCC 25955 and in Citrobacter freundii NCIB 3735. The dehydratases can be resolved by polyacrylamide-gel electrophoresis or separated by anion-exchange chromatography alone. Sucrose density gradient centrifugation failed to distinguish the enzymes and indicated a molecular weight of 1.9 . 10(5) for both. The enzymes can be assayed individually, even when present in the same crude extract, using the 67-fold difference in their Km values for coenzyme B-12. For both enzymes inactivation kinetics are observed with glycerol as substrated, and monovalent cations influence both the inactivation rate and catalytic rate of the reaction.     

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.     

Heterologous expression and characterization of recombinant glycerol dehydratase from Klebsiella pneumoniae in Escherichia coli

Glycerol dehydratase (EC 4.2.1.30), as one of the key enzymes in converting glycerol to the valuable intermediate 1,3-propanediol, is important for biochemical industry. The dhaB genes encoding coenzyme B(12)-dependent glycerol dehydratase in Klebsiella pneumoniae were cloned and expressed in Escherichia coli. An effective co-expression system of multiple subunits protein was constructed. Heterologous expression vectors were constructed using the splicing by overlap extension-PCR technique to co-express the three subunits of the glycerol dehydratase. After induction by isopropyl-beta-D-thiogalactopyranoside, SDS-PAGE analysis revealed that: (i) only the alpha subunit of glycerol dehydratase was expressed in direct expression system, (ii) the three subunits of glycerol dehydratase with predicted molecular massess of 64 (agr;), 22 (beta), and 16 kDa (gamma) were expressed simultaneously in co-expression system, and (iii) the fusion expression system expressed the fusion protein of 99 kDa. Enzyme assay showed that the activities of three heterologous expression products were 27.4, 2.3, and 0.2 U/mg. The highest enzyme activity was almost 17 times of that in K. pneumoniae. The recombinant enzyme was purified and biochemically characterized. The apparent Km values of the enzyme for coenzyme B(12) and 1, 2-propanediol were 8.5 nM and 1.2 mM, respectively. The enzyme showed maximum activity at pH 8.5 and 37 degrees C.     

Nonenzymatic tight binding of radioactivity to macromolecular fractions as a source of error in labeling experiments

A highly sensitive recording method is developed for determining the activity of the CoB-12-dependent enzyme, bacterial glycerol dehydratase. The method is based on the ability of alcohol dehydrogenase, in the presence of NADH₂, to reduce aldehydes formed from glycols by glycerol dehydratase. The rate of the coupled reaction is measured spectrophotometrically by the decrease in optical density at 340 nm or by measuring the decrease of H⁺ concentration with a sensitive pH-metric method. The conditions for coupling these two reactions, method of application, and its advantages and limitations are discussed. The method is highly sensitive and makes it possible to detect 0.5–2.5 × 10^?3 glycerol dehydratase units.     

甘油脱水酶再激活酶的克隆表达及活性鉴定

运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。