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Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

ABSTRACT: Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta(1)-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

SUBMITTER: van Der Geize R

PROVIDER: S-EPMC101450 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

van Der Geize R R Hessels G I GI van Gerwen R R Vrijbloed J W JW van Der Meijden P P Dijkhuizen L L

Applied and environmental microbiology 20000501 5

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of k ...[more]

PMID: 10788377

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Project description:Comamonas testosteroni delta 4(5 alpha)- and delta1-dehydrogenases [delta4(5alpha)- and delta1DH] are key enzymes in the degradation of steroids having an A:B ring fusion in a trans configuration. We previously reported the isolation of the delta1dh gene (P. Plesiat, M. Grandguillot, S. Harayama, S. Vragar, and Y. Michel Briand, J. Bacteriol. 173:7219-7227, 1991). In this study, the gene encoding delta 4(5 alpha)DH was cloned in Escherichia coli on a 16-kbp BamHI fragment by screening a genomic bank of C. testosteroni ATCC 17410 with a probe derived from delta1dh. Subcloning experiments in plasmid pUC19 mapped delta 4(5 alpha)dh immediately downstream of delta1dh. The enzyme was overexpressed 18-fold in cells of E. coli JM109 carrying a 2.5-kbp cloned fragment (plasmid pXE25). However, much higher levels of enzymatic activity (264-fold) were obtained in Pseudomonas putida KT2440, using pMMB208 as an expression vector. Studies with crude lysates of KT2440 showed that delta4(5alpha)DH exhibits higher specificity and higher activity toward delta l-androstene-3,17-dione than toward the saturated derivative 5 alpha-androstane-3,17-dione. The reaction was found to be irreversible and to use efficiently typical flavoprotein electron acceptors; optimal conditions for the enzyme activity were pH 8 and 40 degrees C. Analysis of the nucleotide sequence of the insert of pXE25 revealed an open reading frame of 1,593 bp preceded by a putative ribosome-binding site and followed by a potential transcription terminator. The amino acid sequence of the deduced peptide showed a typical flavin adenine dinucleotide-binding site in its N-terminal region, confirming the flavoproteinic structure of delta 4(5 alpha)DH. The predicted molecular mass was consistent with that of the enzyme expressed in a T7 polymerase system (60 kDa). Alignment between delta 4(5 alpha)dh and delta1dh indicated that both genes, though coding for functionally related enzymes, do not derive from a common ancestor.

Dataset Information

Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

Publications

Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

Similar Datasets

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