Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization

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Abstract
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.