Molecular Cloning, Sequencing and Characterization of the Genes for Adenosylcobalamin-dependent Diol Dehydratase of<i>Klebsiella pneumoniae</i>

Tobimatsu, Takamasa; Azuma, Muneaki; Hayashi, Shigenari; Nishimoto, Kyou Ichi; Toraya, Tetsuo

doi:10.1271/bbb.62.1774

Cited by 17 publications

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“…14,15,23) A comparison of the amino acid sequences of the corresponding subunits between diol and glycerol dehydratases 20,[23][24][25][26][27] showed high identities between them, except that the N-terminal 32 and 37 amino acid residues of the and subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase. Hence it was suggested that these regions might determine the solubility of these enzymes, although the hydropathy plots of diol dehydratase do not show any prominent hydrophobic peaks in these missing regions.…”

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confidence: 99%

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

Tobimatsu

Kawata

Toraya

2005

Bioscience, Biotechnology, and Biochemistry

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Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer ðÞ 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 and subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the and 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 and subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both and subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.

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confidence: 99%

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

Tobimatsu

Kawata

Toraya

2005

Bioscience, Biotechnology, and Biochemistry

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“…In contrast, glycerol dehydratase is highly soluble and exists in a cytosolic fraction. Predicted amino acid sequences of the subunit are well conserved between the two dehydratases except that the ␤ and ␥ subunits of diol dehydratase have extra amino acid residues in their N-terminal regions (16)(17)(18). Although hydropathy profiles show that these N-terminal regions are neither hydrophobic nor hydrophilic, removal of the regions from the ␤ and ␥ subunits of diol dehydratase by limited proteolysis with trypsin (23,34) or by gene engineering techniques (23) makes the enzyme highly soluble.…”

Section: Production and Distribution Of The Hybrid Enzymes Between DImentioning

confidence: 99%

“…1) ( 18 ). The N-terminal 32 and 37 amino acid residues of the ␤ and ␥ subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase (16)(17)(18). These regions of ␤ and ␥ subunits of diol dehydratase lower the solubility of diol dehydratase ( 23 ).…”

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“…[13][14][15]). In order to reveal their similarities and differences on a molecular level, we cloned, sequenced, and expressed the diol dehydratase genes of Klebsiella oxytoca ( 16 ) and K lebsiella pneumoniae ( 17 ) and the glycerol dehydratase genes of K. pneumoniae ( 18 ). The glycerol dehydratase genes of Citrobacter freundii ( 19 ) and Clostridium pasteurianum ( 20 ) and the diol dehydratase genes of Salmonella typhimurium ( 21 ) and Lactobacillus collinoides ( 22 ) have also been cloned by other investigators.…”

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Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

Sakai

Yamasaki

Toyofuku

et al. 2007

J Nutr Sci Vitaminol

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Summary Adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase are isofunctional enzymes that catalyze the dehydration of 1,2-diols to the corresponding aldehydes. Although they bear different metabolic roles, both enzymes consist of three different subunits and possess a common ( ␣␤␥ ) 2 structure. To elucidate the roles of each subunit, we constructed expression plasmids for the hybrid dehydratases between diol dehydratase of Klebsiella oxytoca and glycerol dehydratase of Klebsiella pneumoniae in all the combinations of subunits by gene engineering techniques. All of the hybrid enzymes were produced in Escherichia coli at high levels, but only two hybrid enzymes consisting of the ␣ subunit from glycerol dehydratase and the ␤ subunits from diol dehydratase showed high activity. The substrate specificity, the susceptibility to inactivation by glycerol, and the monovalent cation specificity of the wild type and hybrid enzymes were primarily determined by the origin of their ␣ subunits. Key Words coenzyme B 12 , adenosylcobalamin, diol dehydratase, glycerol dehydratase, hybrid enzymes Diol dehydratase ( DL -1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (EC 4.2.1.30) are isofunctional enzymes that catalyze the AdoCbl 1 -dependent conversion of 1,2-diols to the corresponding deoxy aldehydes ( 1-3 ). They consist of the three different subunits, ␣ ( Mr 60,000-61,000), ␤ ( Mr 21,000-24,000), and ␥ ( Mr 16,000-19,000), and dissociate into two dissimilar protein components in the absence of substrate ( 4, 5 ). Large and small components correspond to the ␣ 2 ␥ 2 complex and the ␤ subunit, respectively ( 6, 7 ). Their catalytic properties are similar, but they are different in the binding affinity for AdoCbl ( 8,9 ), substrate specificity ( 1-3, 10, 11 ), susceptibility to suicide inactivation by glycerol ( 2, 9, 10 ), monovalent cation specificity ( 1,11,12 ), and immunochemical reactivity toward anti-diol dehydratase antiserum ( 11 ) (for review, see Refs. 13-15 )). In order to reveal their similarities and differences on a molecular level, we cloned, sequenced, and expressed the diol dehydratase genes of Klebsiella oxytoca ( 16 ) and K lebsiella pneumoniae ( 17 ) and the glycerol dehydratase genes of K. pneumoniae ( 18 ). The glycerol dehydratase genes of Citrobacter freundii ( 19 ) and Clostridium pasteurianum ( 20 ) and the diol dehydratase genes of Salmonella typhimurium ( 21 ) and Lactobacillus collinoides ( 22 ) have also been cloned by other investigators. Comparison of the deduced amino acid sequences of the corresponding subunits between diol and glycerol dehydratases indicated that the ␣ subunit is most highly conserved among the subunits of the enzymes (identity, 71%), whereas the homologies of ␤ and ␥ subunits are lower (identities, 58% and 54%, respectively) ( Fig. 1) ( 18 ). The N-terminal 32 and 37 amino acid residues of the ␤ and ␥ subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase (16)(17)(18). These regions ...

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“…Several of the glycerol fermentation genes, which form the dha regulon, have been cloned and sequenced (11,(41)(42)(43).…”

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Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae : Evidence for Involvement of the Cyclic AMP Receptor Protein

2001

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Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na ؉ -dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomal citS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of K. pneumoniae bound to two sites in the citC-citS intergenic region, which were centered at position ؊41.5 upstream of the citC and citS transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.Klebsiella pneumoniae, a member of the Enterobacteriaceae, is able to grow with citrate as a sole carbon and energy source under anoxic conditions (for a review, see reference 4). Citrate fermentation involves uptake by a Na ϩ -dependent citrate carrier (32, 44), cleavage into acetate and oxaloacetate by citrate lyase, and decarboxylation of oxaloacetate to pyruvate by the Na ϩ ion pump oxaloacetate decarboxylase (12, 13). The conversion of pyruvate to acetate, formate, CO 2 , and H 2 (7, 38) is catalyzed by enzymes generally involved in anaerobic pyruvate catabolism. The citrate-specific fermentation genes form a cluster of two divergent operons (5, 6). The citCDEFG operon encodes citrate lyase ligase (CitC), the citrate lyase subunits (CitD, CitE, and CitF), and triphosphoribosyl-dephospho-coenzyme A synthase (CitG), which catalyzes the formation of a precursor of the citrate lyase prosthetic group (36, 37). The citS-oadGAB-citAB operon encodes the citrate carrier CitS, the oxaloacetate decarboxylase subunits (OadG, OadA, and OadB), and a two-component system composed of the sensor kinase CitA and the response regulator CitB (Fig. 1A). The CitA-CitB system was shown to be essential for the expression of both operons (6). CitA presumably functions as a sensor of extracellular citrate, since the periplasmic domain of this membrane-bound protein binds citrate with high affinity (K d Ϸ 6 M) and specificity (23). CitB acts as a transcriptional activator of the citS and the citC operons and binds to two sites in the 193-bp citC-citS intergenic region (Fig. 1) in a phosphorylation-dependent fashion (24).Maximal expression of a chromosomally integrated translational citSЈ-ЈlacZ fusion requires citrate, anoxic...

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Molecular Cloning, Sequencing and Characterization of the Genes for Adenosylcobalamin-dependent Diol Dehydratase ofKlebsiella pneumoniae

Cited by 17 publications

References 13 publications

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae : Evidence for Involvement of the Cyclic AMP Receptor Protein

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