title: Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.
abstract: In order to assess the roles of specific amino acid residues in the delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B during catalysis, we replaced aspartic acid 40 with asparagine (D40N) and tyrosine 16 with phenylalanine (Y16F) in the enzyme by site-directed mutagenesis. Both purified mutant enzymes resulted in profound decreases in catalytic activities, 10(3.3)-fold in the Y16F mutant and 10(6.2)-fold in the D40N mutant. Aspartic acid 40 and tyrosine 16 of the enzyme are the corresponding amino acids in the active site of the homologous enzyme from Comamonas testosteroni. Our results indicate that active-site residues of the two homologous enzymes are similar. This is opposite to the previous identification of a cysteine in an active site-directed photoinactivation study of the enzyme.
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology, Kyungbuk, Republic of Korea.
authors.forename: S W
authors.initials: SW
authors.lastname: Kim
authors.forename: K Y
authors.initials: KY
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence; D000735:Androstenedione / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013251:Steroid Isomerases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D000735:Androstenedione; C003528:5-androstene-3,17-dione; D013251:Steroid Isomerases; C022660:steroid delta-isomerase