Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase

Mallinder, Phillip R.; Pritchard, A.M.; Moir, Anne

doi:10.1016/0378-1119(92)90438-u

Cited by 29 publications

(16 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The dhaD gene product showed a high degree of homology to the glycerol dehydrogenase (gldA) of E. coli (3) and Bacillus stearothermophilus (26), the fraction of identical amino acids being 57.4 and 49.6%, respectively. Lower homology values (21.0 to 23.7% identity, 44.3 to 50.2% similarity) to a novel family of alcohol dehydrogenases (type III) were obtained, including, e.g., Adh2 of Zymomonas mobilis (8) and AdhE of E. coli (17; for a review, see reference 33).…”

Section: ϩmentioning

confidence: 99%

Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii

1995

View full text Add to dashboard Cite

Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent K m s of the enzyme for glycerol and NAD ؉ were 1.27 mM and 57 M, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent K m s were 30 and 70 M, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the 54 bacterial enhancer-binding protein family.Microorganisms such as Citrobacter freundii and Klebsiella pneumoniae are able to grow anaerobically on glycerol as the sole carbon and energy source (19). In the absence of an external oxidant, glycerol is fermented by a dismutation process involving two pathways, one serving for glycerol oxidation and the other for the consumption of the reducing equivalents generated. The oxidation of glycerol is catalyzed by NAD ϩ -linked glycerol dehydrogenase, which converts the substrate to dihydroxyacetone (DHA). This product is then phosphorylated by DHA kinase and funneled to glycolysis (15). Generation of NAD ϩ is achieved by the sequential action of coenzyme B 12 -dependent glycerol dehydratase and NADH-linked 1,3-propanediol dehydrogenase (15). Glycerol is first converted to 3-hydroxypropionaldehyde, which then is reduced to 1,3-propanediol, accounting for about 50 to 66% of the glycerol consumed. The four key enzymes of this pathway are encoded by the dha regulon, the expression of which is induced when DHA or glycerol is present (15, 34). The entire dha regulon was cloned from C. freundii (10) and K. pneumoniae (48), but molecular data are available only for C. freundii.Recently we have subcloned, sequenced, and overexpressed the gene encoding 1,3-propanediol dehydrogenase (9). In this report, we describe the purification of glycerol dehydrogenase (EC 1.1.1.6) and DHA kinase (EC 2.7.1.29) from C. freundii and the cloning, identification, and organization of the corresponding genes. MATERIALS AND METHODSMaterials. Q-Sepharose Fast Flow, Reactive Red, and Blue Sepharose CL-6B were obtained from Pharmacia LKB GmbH (Freiburg, Germany), and hydroxyapatite was from Sigma Chemie (Deisenhofen, Germany). Tris, EDTA, and sodium dodecyl sulfate were from...

show abstract

Section: ϩmentioning

confidence: 99%

Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii

1995

View full text Add to dashboard Cite

show abstract

“…Each of the ORFs that flank mip has been named on the basis of their similarity to known E. coli gene products (Bachmann, 1990). The predicted product of the partial C. psittaci asps exhibited 52% identity with the last 98 amino acids of aspartate tRNA synthase from E. coli (Sharples & Lloyd, 1991), the predicted product of C. psittaci spoU exhibited 39% identity with the SpoU homologue from E. coli (Koonin & Rudd, 1993;Mallinder et al, 1992) oligonucleotide pairs (Table 1) were used to amplify homologous sequences from genomic DNAs of B577 and…”

Section: Nucleotide Sequence Analysis Of the Genomic Dna Flanking Mipmentioning

confidence: 99%

A 28 kDa major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp. and Chlamydia trachomatis - cloning and characterization of the C. psittaci mip-like gene

et al. 1996

View full text Add to dashboard Cite

“…39 kDa, which distinguishes it from the other cis-diol dehydrogenases reported to date (33,35,40). In addition, both the nucleotide sequence and predicted 365-amino-acid sequence exhibit significant homology to sequences of glycerol dehydrogenases from Escherichia coli (6), Citrobacter freundii (9), and Bacillus stearothermophilus (30), which are members of the type III alcohol dehydrogenase family.…”

mentioning

confidence: 94%

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase

1996

View full text Add to dashboard Cite

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD ؉ -dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD was overexpressed in Escherichia coli and purified. It was found to utilize NAD ؉ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.A wide variety of natural and synthetic aromatic compounds are metabolized by many soil bacteria, notably Pseudomonas species. In the aerobic degradation of these aromatic compounds, reactions of metabolic pathways generally lead to the formation of dihydroxy aromatic intermediates such as catechols. The formation of these intermediates is carried out by two successive enzymatic steps. The initial dihydroxylation step is carried out by a ring-hydroxylating dioxygenase enzyme that incorporates both atoms of dioxygen into the aromatic nucleus to form cis-dihydrodiols. This is followed by a dehydrogenation reaction catalyzed by a cis-dihydrodiol dehydrogenase to give catechols (18). The ring-hydroxylating dioxygenases are multicomponent in nature, comprising an electron transport chain and a terminal catalytic component (3,13,45,47). By contrast, almost all of the cis-dihydrodiol dehydrogenases reported so far are homotetramers of a 28-kDa polypeptide and are similar with regard to their specificity for cis-dihydrodiols and their absolute requirement for NAD ϩ as their primary electron acceptor (33,35,36,40). Further catabolism of catechols involves cleavage of the ring in either an ortho (intradiol) or a meta (extradiol) manner, with the products of ring cleavage being channeled into the tricarboxylic acid cycle (8).Pseudomonas putida ML2 (NCIB 12190) is able to utilize benzene as a sole source of carbon and energy through the ortho pathway (Fig. 1) (2). Benzene dioxygenase, a soluble multicomponent...

show abstract

Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase

Cited by 29 publications

References 22 publications

Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii

Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii

A 28 kDa major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp. and Chlamydia trachomatis - cloning and characterization of the C. psittaci mip-like gene

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase

Contact Info

Product

Resources

About