Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3α/β,20β-hydroxysteroid dehydrogenase

Abstract

Pig 3α/β,20β-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. 3α/β,20β-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 20β-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 3α/β,20β-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 3α/β,20β-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.