The purification and characterization of delta 5-3-ketosteroid isomerase from Pseudomonas putida, a cysteine-containing isomerase.
A delta 5-3-ketosteroid isomerase (EC 5.3.3.1) has been isolated from Pseudomonas putida Biotype B and purified to homogeneity. This previously undescribed steroid isomerase resembles that isolated from Pseudomonas testosteroni (Talalay, P., and Wang, V.S. (1955) Biochim. Biophys. Acta 18, 300-301). The enzyme is induced by various steroids, has a subunit molecular weight of 13,750 +/- 250, a pI of 4.8 +/- 0.1 has a specific activity of 40,000 units/mg, using 5-androstene-3,17-dione as the substrate. The amino acid composition of the enzyme subunit is Lys 2, His 2, Arg 8, Asp 11, Thr 5, Ser 4, Glu 17, Pro 8, Gly 12, Ala 12, Val 10, Met 5, Ile 6, Leu 8, Tyr 4, Phe 4, and Cys 4. The amino acid sequence has been determined for the NH2-terminal 50 residues. This portion of the polypeptide chain is approximately 47% homologous with the amino acid sequence of the first 50 residues of the delta 5-3-ketosteroid isomerase from P. testosteroni. The amino acid sequence of residues 33 to 41 of the P. putida isomerase is identical with the region Ala 31 through Pro 39 in the P. testosteroni enzyme. Residue 40 of the P. putida enzyme is aspartic acid and corresponds in the sequence to Asp 38 of the P. testosteroni isomerase, which has been shown to be essential for enzymatic activity (Ogez, J.R., Tivol, W.F., and Benisek, W.F. (1977) J. Biol. Chem. 252, 6151-6155).
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003545:Cysteine / Q000032:analysis*; D007535:Isomerases / Q000302:isolation & purification; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013045:Species Specificity; D013251:Steroid Isomerases / Q000302:isolation & purification
D000596:Amino Acids; D007535:Isomerases; D013251:Steroid Isomerases; D003545:Cysteine