Identification and isolation of glucose dehydrogenase genes of <i>Bacillus megaterium</i> M1286 and their expression in <i>Escherichia coli</i>

Heilmann, Hans J.; Mägert, Hans J.; Gassen, Hans Günter

doi:10.1111/j.1432-1033.1988.tb14124.x

Cited by 52 publications

(33 citation statements)

References 26 publications

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“…4). GNO from G. oxydans displayed the typical pattern of six strictly conserved residues which has been found in all group II alcohol dehydrogenases that have been sequenced, i.e., glucose dehydrogenase from Bacillus megaterium (18), alcohol dehydrogenase from Drosophila melanogaster (45), ribitol dehydrogenase from Klebsiella aerogenes (13), 3-␤-hydroxysteroid dehydrogenase from Pseudomonas testosteroni (51), cis-benzene glycol dehydrogenase from Pseudomonas putida (19), and acetoacetyl-coenzyme A reductase from Alcaligenes eutrophus (29). The glycine residues at positions 18, 22, and 24 correspond to the spacing expected for pyridine nucleotide binding sites, as shown for other dehydrogenases (20,33,49).…”

Section: Resultsmentioning

confidence: 85%

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans

1995

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Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent K m values were 20.6 mM for the substrate gluconate and 73 M for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa. Plasmidencoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E. coli and with 6.80 U/mg of cell-free protein in G. oxydans, which corresponded to 85-fold overexpression of the G. oxydans wild-type GNO activity. Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.Gluconobacter oxydans, a gram-negative, strictly aerobic, rod-shaped bacterium, belongs to the family of acetic acid bacteria (11) and oxidizes glucose via two alternative pathways (28): one requires an initial phosphorylation followed by oxidation via the hexose monophosphate pathway (28); the second involves the nonphosphorylative formation of gluconic acid and of ketogluconic acids (12,22). The enzyme catalyzing the formation of gluconic acid, glucose dehydrogenase, is located in the cytoplasmic membrane of G. oxydans and is coupled to the respiratory chain (3). Further oxidation of gluconic acid by the membrane-bound and respiratory chain-coupled enzymes gluconate dehydrogenase and 2-ketogluconate dehydrogenase leads to the formation of 2-ketogluconic and 2,5-diketogluconic acids (39, 40). The enzymatic activity converting gluconic acid to 5-ketogluconic acid, however, is located in the cytoplasm of G. oxydans and depends on the presence of the cofactor NADP (12, 27). The enzymes catalyzing this reaction have been purified from G. suboxydans and Gluconobacter liquefaciens (1, 2). However, these enzymes were termed 5-ketogluconate...

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Section: Resultsmentioning

confidence: 85%

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans

1995

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“…9) revealed that all of these residues except tyrosine were located at the N-terminal ends of putative p-pleated sheets, the latter being located at the carboxyl end of a ,B sheet. (20), ribitol dehydrogenase from Klebsiella aerogenes (13), and alcohol dehydrogenase from D. melanogaster (44). Multiple sequence alignment and gapping was done by using the GCG Gap, Lineup, and Pretty programs, and a consensus sequence was generated.…”

Section: Resultsmentioning

confidence: 99%

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708

1991

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Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7a-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gtll, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7a-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7a-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5a transformant harboring pBH51 had approximately 30-fold greater levels of 7a-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7af-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reacted with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7a-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding.Many members of the human intestinal microflora can modify the glycine and taurine conjugates of the primary bile acids, cholic acid and chenodeoxycholic acid, producing up to 20 different bile acid metabolites (21). The predominant modifications include deconjugation of the glycine and taurine moieties by bile salt hydrolase, removal of 7-hydroxy groups (7-dehydroxylation), and dehydrogenation of a-and ,B-hydroxy groups at carbons 3, 7, and 12 by stereospecific bile acid hydroxysteroid dehydrogenases to yield oxo bile acids.Quantitatively, the most important of these transformations is the 7ux-dehydroxylation of cholic and chenodeoxycholic acids, producing deoxycholic and lithocholic acids, respectively (43). The human intestinal anaerobic bacterium, Eubacterium sp. strain VPI 12708, has a multistep 7a-dehydroxylation pathway which is induced by addition of cholic acid to the culture medium (6,9,11, 22,(46)(47)(48). Several bile acid-inducible (bai) genes encoding proteins involved in this pathway have been cloned and sequenced (10,11,18,31,49,50).7a-Hydroxysteroid dehydrogenase is the most common class of bile acid hydroxysteroid dehydrogenases found in nature (3), having been detected in numerous genera of bacteria (3, 14-16, 28, 30, 37) and in mammalian liver (1). 7a-Hydroxysteroid dehydrogenases have been purified from Bacteroides species (23,29,41), Escherichia coli (28,36,37), and rat liver microsomes (1). Recently, two 7a-hydroxysteroid dehydrogenases have been pu...

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“…The ORF4 product is similar to a group of oxidoreductases ( Fig. 3), including the protochlorophyllide reductase (Pcr) of barley (33), the glucose dehydrogenase of Bacillus megateium (19), the ribitol dehydrogenase of Kgebsiella aerogenes (28), and the NodG protein of Rhizobium meliloti (which is homologous to ,B-ketoacyl reductase [34]). The similarity among these oxidoreductases was stronger at the N termini.…”

Section: Methodsmentioning

confidence: 99%

Discovery and characterization of a new transposable element, Tn4811, in Streptomyces lividans 66

Chen¹,

Yu²,

Chung³

et al. 1992

J Bacteriol

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Transposition of a new 5.4-kb transposon, Tn4811, of Streptomyces lividans to the melC operon of Streptomyces antibioticus on plasmid pU702 was discovered. The nucleotide sequence of this copy of Tn4811, which contained an imperfect (9 of 11 bp) terminal inverted repeat, five putative Streptomyces coding sequences for an oxidoreductase and its transcription regulator, and three transposition-related proteins, was determined. SLP-strains of S. lividans contained one copy (A) of Tn4811, while SLP2+ strains contained an additional copy (B) on the SLP2 plasmid. The nucleotide sequences at three insertion junctions of Tn4811 were determined. Copy B lacked 41 bp from the left end. At the other five junctions the duplication of a putative 3-bp target sequence (TGA) was observed. A sequence of less than 3 kb homologous to Tn4811 was present in S. antibioticus. DNA homologous to Tn4811 was not detected in 14 other Streptomyces species.Structural instability of genomic DNA is a widespread feature of gram-positive, filamentous, soil bacteria of the genus Streptomyces (32). Many genetic traits undergo spontaneous loss in laboratory cultures at frequencies of 10' to 10-2. Exposure to certain stresses such as UV irradiation, DNA intercalating agents, cold temperature, or protoplasting and regeneration increases the frequency of these instabilities. The mutations involved in the instabilities are attributed to large deletions of chromosomal DNA, which are frequently accompanied by tandem amplifications of particular sequences nearby (32). The genetic principle(s) underlying the structural instability is not clear. Possible mechanisms involved are homologous recombination, site-specific recombination, and transposition. Homologous recombination systems in Streptomyces species and their contribution to genetic instability have received little study. Tsai and Chen (41) isolated a rec mutant defective in intraplasmid recombination but not in chromosomal recombination (25). Chou and Chen (11), in their investigation of an unstable arg gene, found no effect of this rec mutation on its instability. Site-specific recombination and transposition, although representing forms of fluidity of genomic DNA, have not been implicated in the instability of other DNA sequences (8).About one-third of Streptomyces species can produce melanin pigment (44). The melanin (melC) operon of Streptomyces antibioticus has been cloned (24), sequenced (3), and widely used in many recombinant vectors. The melC operon in the Streptomyces species examined is genetically unstable, undergoing spontaneous deletions at relatively high frequencies-about 10-' (32). Similar to those observed in other unstable genes in Streptomyces species, the deletions of melC were frequently accompanied by extensive tandem amplifications of specific sequences (18,32).

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Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli

Cited by 52 publications

References 26 publications

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708

Discovery and characterization of a new transposable element, Tn4811, in Streptomyces lividans 66

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