Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli.
The intestinal microorganism Eubacterium sp. VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7 alpha-dehydroxylation of cholic acid to deoxycholic acid. The baiH gene encoding NADH:FOR was subcloned into an IPTG-inducible expression vector, pBaiH2.2. Escherichia coli DH5 alpha cells transformed with pBaiH2.2 expressed 10-fold higher levels of NADH:FOR upon induction with IPTG than did Eubacterium sp. VPI 12708 cells induced with cholic acid. The NADH:FOR produced by E. coli DH5 alpha(pBaiH2.2) was purified to > 95% electrophoretic homogeneity in three steps. The purified NADH:FOR was similar to that of Eubacterium sp. VPI 12708 in subunit and native M(r) (ca. 72,000 and 210,000, respectively), pH optimum, sensitivity to inhibitors, and electron acceptor specificity. It contained 1 mol of FAD, up to 2 mol of iron, and 1 mol of copper per mol of subunit. The enzyme reduced synthetic quinones, dyes, flavins, and O2 with NADH as the electron donor, but did not reduce disulfide compounds, various unsaturated bile acids, cytochrome c, physiological quinones, or cell fractions from Eubacterium sp. VPI 12708. Addition of purified NADH:FOR to Eubacterium sp. VPI 12708 cell extracts altered the balance of oxidized and reduced bile acid intermediates produced during cholic acid 7 alpha-dehydroxylation, suggesting that the enzyme may regulate the cellular ratio of NAD to NADH.
Baron
S F
SF
Department of Microbiology and Immunology, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298-0678, USA.
Hylemon
P B
PB
D000595:Amino Acid Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology; D003001:Cloning, Molecular; D003300:Copper / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D005051:Eubacterium / Q000235:genetics*; D038181:FMN Reductase; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis
D001647:Bile Acids and Salts; D011994:Recombinant Proteins; D009243:NAD; D003300:Copper; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases