Defense Date

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Phillip B. Hylemon

Abstract

Clostridium scindens is an obligate anaerobe isolated from the normal intestinal flora of man. It is the only bacterium known at present to synthesize both steroid-17,20-desmolase and 20α-hydroxysteroid dehydrogenase (ZOα-HSDH). This study was undertaken to characterize these neutral steroid transforming reactions in extracts of C. scindens; to purify and characterize the enzymes responsible for side-chain cleavage and 20α- hydroxysteroid oxidoreduction and to clone the genes encoding these enzymes with a view towards studying the mechanism of induction by steroids in a prokaryotic system. Both enzymes were found to be co-inducible in cells cultured in the presence of 17α-hydroxysteroids. In cell extracts, only 17α,21- dihydroxysteroids served as substrates for both activities. Desmolase has been partially purified by conventional DEAE- cellulose chromatography. Although the desmolase reaction was stimulated by NAD+ and bivalent metal cations in cell extracts, the partially purified enzyme was only reactive with coenzyme B12. The observed difference in cofactor requirements in crude cell extracts and partially purified preparations is not understood at present. 20α-HSDH has been purified 252-fold to apparent homogeneity by a method involving ammonium sulfate fractionation, conventional DEAE-cellulose chromatography, elution from Cibacron blue agarose, gel filtration HPLC and DEAE HPLC. The pH optimum for reduction of the 20-ketone of cortisol with NADH is between pH 6-6.5. The pH optimum for oxidation of the 20α-hydroxyl of 20α-dihydrocortisol with NAD+ is approx. pH 8. Michaelis constants were determined: Km for NADH (8 μM); cortisol (32 μM); cortisone (22μM); NAD+ (526μM): 20α-dihydrocortisol (41 μM). The native enzyme is apparently a tetramer comprised of subunits with a Mr 40,000. Although two bands with a slightly different charge (pI approx. 6.1) were observed on two-dimensional PAGE, a single N-terminus was obtained by gas phase sequencing. A computer aided search (FASP) showed that the partial amino acid sequence (1-11) was highly homologous with glyceraldehyde-3-phosphate dehydrogenases (GAPDH) isolated from a variety of sources. Residues 4-8 of GAPDH are involved in binding of the pyridine nucleotide cofactor. On Western blots of cortisol induced and uninduced cell extracts, two bands differing in size by approximately 2000 daltons were observed. The presence of immunoreactive proteins with the same molecular weight as the purified 20α-HSDH in uninduced cell extracts may be due to cross-reactivity with constitutive GAPDH (Mr, 39,000). Using a 32P-labelled oligonucleotide probe corresponding to amino acids 3-8, two discrete bands were seen on Southern blots of EcoR1 digests of C. scindens genomic DNA. Efforts to clone these fragments in lambda gt11 were unsuccessful.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-1-2017

Included in

Microbiology Commons

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