Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Abstract

The structural gene coding for the A5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. pui biotype B genomic library in Xgtll. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986 A5-3-Ketosteroid isomerase (KSI) catalyzes the allylic isomerization of the 5,6 double bond of A5-3-ketosteroids to the 4,5 position by stereospecific intramolecular transfer of a proton (1) (Fig. 1) testosteroni KSI structure has been studied by X-ray crystallography. Three-dimensional structure of the KSI is known at 6-A (0.6-nm; 1 A = 0.1 nm) resolution (21). A preliminary, unrefined model has been obtained on the basis of a 2.5-A resolution electron density map (9). However, this model possesses an unacceptably high R-factor, and efforts to refine this structure in order to lower the R-factor have not been successful (hla). Part of the difficulty in solving the structure of C. testosteroni KSI arises from the very large size and anisotropy of the unit cell [P6 (1) The polypeptide of the KSI from P. putida biotype B is 6 residues longer than that of C. testosteroni KSI. As aligned, there are 44 identical matches, giving only 34% identity between two KSIs (11). A computer search of the sequence databases for proteins homologous to P. putida KSI has yielded no similar proteins other than C. testosteroni KSI. The critical active-site residues of C. testosteroni KSI, Asp-38 and Tyr-14, are conserved in the P. putida KSI polypeptide as Asp-40 and Tyr-16, respectively (reference 11 and this work).The P. putida KSI contains three cysteines and two tryptophans, whereas the C. testosteroni KSI lacks these amino acids.The P. putida KSI showed dependencies of Vm. and Km on pH that differ from those of C. testosteroni KSI (19). An activesite-directed photoinactivation study of the P. putida KSI implicated the modification of the sulfhydryl group in a cysteine, the extent of which correlated with the observed loss 6672 Vol. 21,No. 11

Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

“…The amino acid sequence of the enzyme has been confirmed by the nucleotide sequence of the gene encoding the isomerase which was isolated from P. putida biotype B (10). The polypeptide of the P. putida isomerase is six residues longer than that of the C. testosteroni isomerase and contains three cysteines and two tryptophans which are lacking in the C. testosteroni isomerase.…”

“…Even though there are 44 identical matches giving only 34% identity between the amino acid sequences of the two isomerases, both enzymes have an extensive homology in the amino acid sequence of the amino-terminal region (17). The critical active site residues of the C. testosteroni KSI, Asp-38 and Tyr-14, are well conserved at Asp-40 and Tyr-16 in the P. putida KSI polypeptide, respectively (10,17). In spite of strong homology in the putative active site regions of both isomerases, an active site-directed photoinactivation study of the P. putida isomerase implicated the modification of a cysteine, the extent of which correlated with the observed loss of enzyme activity (22).…”

“…Boxes indicate the amino acids targeted for site-specific mutagenesis. Sequences are derived from nucleotide sequences of C. testosteroni (5, 13) and of P. putida (10).…”

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

“…The EcoRI-HindIII fragments containing the ksi gene in the recombinant plasmid (pKK-KSI) expressing KSI (15) were ligated into the previously digested pBluescript SK(Ϫ) vector (Stratagene) to make pSK-KSI. The mutagenesis was done as described previously (14) by using uracil-containing single-stranded pSK-KSI DNA as the template and the synthetic oligonucleotide as the primer ( Table 1).…”

“…The cloning and sequencing of the gene encoding KSI from P. putida biotype B and its subsequent overexpression have provided an alternative opportunity to study KSI (15). This dimeric enzyme consists of two identical polypeptide chains of 131 amino acids each with a calculated molecular weight of 14,536.…”