Cloning and characterization of the NAD-dependent 7alpha-Hydroxysteroid dehydrogenase from Bacteroides fragilis.
The NAD-linked 7alpha-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was characterized and its gene cloned. The enzyme displayed optimal activities at pH 8.5 (NAD reduction) and 6.5 (NADH oxidation). The lowest K(m) and highest V(max) values were observed with chenodeoxycholic acid and its conjugates. The protein had subunits of 27.4 kDa and a native size of 110 kDa, suggesting a homotetrameric composition. The enzyme was relatively thermostable, retaining 95% of initial activity after 1 h at 65 degrees C. A DNA probe based on the N-terminal amino acid sequence hybridized to a 2373-bp HindIII fragment of B. fragilis DNA. This fragment was cloned into E. coli and sequenced, revealing a 780-bp open reading frame. The predicted amino acid sequence of the ORF showed strong sequence similarity to three other bacterial 7-HSDHs, all in the short-chain dehydrogenase family. The regulation of expression of this gene is currently under investigation.
Bennett
Michael J
MJ
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA.
McKnight
Susan L
SL
Coleman
James P
JP
D000595:Amino Acid Sequence; D001441:Bacteroides fragilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000737:chemistry; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D002635:Chenodeoxycholic Acid; D009243:NAD; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase