DOI
https://doi.org/10.25772/P6WZ-5X22
Defense Date
1990
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Microbiology & Immunology
First Advisor
Phillip B. Hylemon
Abstract
Two enzymes, a 7α-hydroxysteroid dehydrogenase (7α-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 7α-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 7α-HSDH was able to utilize a variety of bile acids containing an unhindered 7α-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 7α-HSDH was cloned on a 3.8 Kb Kpnl-Pstl 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 transcriptional 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 7α-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 5OO-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 α/β unsaturated carboxylates, this homology may be indicative of the physiological function of the NADH:FOR in Eu. sp VPI 12708.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-8-2018
Comments
Scanned, with permission from the author, from the original print version, which resides in University Archives.