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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Kim SW,Choi KY

doi

10.1128/jb.177.9.2602-2605.1995

subject

Has Abstract

pub_date

1995-05-01 00:00:00

pages

2602-5

issue

9

eissn

0021-9193

issn

1098-5530

journal_volume

177

pub_type

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