Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli

Abstract

The intestinal microorganism Eubacterium sp. VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7α-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 DH5α 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 DH5α(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 O z 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 7α-dehydroxylation, suggesting that the enzyme may regulate the cellular ration of NAD to NADH.