Overview

• TITLE – Purification and characterization of two oxidoreductases involved in bile acid modification by the intestinal anaerobe Eubacterium sp. VPI 12708.

• INSTITUTION – Medical College of Virginia / Virginia Commonwealth University

• ADVISOR – Dr. Phillip B. Hylemon

• DATE – 1990

Abstract

Two enzymes, a 7$\alpha$-hydroxysteroid dehydrogenase (7$\alpha$-HSDH) and an NADH-dependent flavin oxidoreductase (NADH:FOR), have been purified to apparent electrophoretic homogeneity from the intestinal anaerobe Eubacterium sp. VPI 12708. Using a protocol consisting of four chromatographic separations, the 7$\alpha$-HSDH was purified by a factor of over 1200-fold with more than a 30% final recovery. Subunit molecular mass was estimated to be 32 Kdal by SDS-PAGE, while native molecular mass estimates from gel filtration were 124 Kdal. The purified 7$\alpha$-HSDH was able to utilize a variety of bile acids containing an unhindered 7$\alpha$-hydroxy moiety as substrates, existing either as free acids or glycine or taurine conjugates. The presence of an oxo moiety at position 3 or 12 profoundly altered the kinetic values for this enzyme. The structural gene for the 7$\alpha$-HSDH was cloned on a 3.8 Kb Kpn I - Pst I fragment and was sequenced using the dideoxy chain termination method. An open reading frame of 798 bp encoding a 266 amino acid protein was detected. The N-terminal amino acid sequence of the purified protein was identical to the first 22 amino acids predicted from the open reading frame. Putative transciptional promotor and terminator regions along with a tentative ribosome binding site were also located. Northern blot analysis indicated that this protein was expressed constitutively on an approximately 1 Kb monocistronic message. During sequence analysis, the 7$\alpha$-HSDH was found to be highly homologous to several members of the short-chain, non-zinc alcohol/polyol dehydrogenase superfamily. Using a five step protocol, the NADH:FOR was also purified to homogeneity. A final purification of greater than 500-fold with an 11% recovery was obtained. The purified protein had a subunit molecular mass of 72 Kdal and a native mass of 210 Kdal, suggesting that it exists either as a dimer or a trimer. Northern blot, Western blot, and activity stains of native gels all indicated that the NADH:FOR is a cholate-inducible protein. N-terminal amino acid sequence determination revealed a significant homology to enoate reductase from Clostridium kluyveri. Since the enoate reductase is involved in the reduction of a variety of $\alpha / \beta$ unsaturated carboxylates, this homology may be indicative of the physiological function of the NADH:FOR in Eu. sp VPI 12708.

Dissertation Full Text

The full text of this dissertation is available in the VCU Scholars Compass as a PDF file. I have also posted a copy of the thesis on the site here.