3-KETOSTEROID-DELTA(1)-DEHYDROGENASE OF RHODOCOCCUS-RHODOCHROUS - SEQUENCING OF THE GENOMIC DNA AND HYPEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

The gene encoding 3-ketosteroid-Delta(1)-dehydrogenase from Rhadococcu s rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1, 536 nucleotides and encodes an enzyme protein of 511 amino acid residu es. The amino terminal methionine residue was deleted in the mature pr otein. The amino acids involved in the flavin binding site are conserv ed in the dehydrogenase sequence. The deduced amino acid sequence is h ighly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a hea t shock protein gene, dnaJ, and downstream, a gene of a hypothetical p rotein. The enzyme gene was ligated with an expression vector to const ruct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta(1)-dehydrogen ase as an active and soluble protein at more than 30 times the level o f R. rhodochrous cells. Purification of the recombinant 3-ketosteroid- Delta(1)-dehydrogenase from. the E, coli cells by a simplified procedu re yielded about 13 mg of enzyme protein/liter of the bacterial cultur e. The purified recombinant dehydrogenase exhibited identical molecula r and catalytic properties to the R, rhodochrous enzyme.