Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase.
Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3alpha/beta,20beta-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20beta-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4-1% and the mutant with arginine-198 has a catalytic efficiency of 4-23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3alpha/beta,20beta-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.
Nakajin
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
Takase
N
N
Ohno
S
S
Toyoshima
S
S
Baker
M E
ME
D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D014443:Tyrosine; D015089:20-Hydroxysteroid Dehydrogenases; C115140:3 alpha-beta, 20 beta-hydroxysteroid dehydrogenase; D008239:Lysine