Author/Editor		Kristan, Katja; Stojan, Jure; Moeller, Gabriele; Adamski, Jerzy; Lanišnik-Rižner, Tea
Title		Coenzyme specificity in fungal 17beta-hydroxysteroid dehydrogenase
Type		članek
Source		Mol Cell Endocrinol
Vol. and No.		Letnik 241
Publication year		2005
Volume		str. 80-7
Language		eng
Abstract		The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus is an NADP(H)-dependent member of the short-chain dehydrogenase/reductase superfamily (SDR) that belongs to the cP1 classical subfamily. Here, we have created several mutants by sitedirected mutagenesis, and through these we have studied the amino acid residues that are responsible for coenzyme binding and specificity. The Thr202Val and Thr202Ile mutants were inactive, thus confirming the importance ofThr202 for the appropriate orientation ofthe coenzyme that enables the hydride transfer. The Ala50Arg and Asn51 Arg mutants had increased rates of NADPH dissociation, and thus an enhanced substrate oxidation with NADP+, while the Asn51Arg mutant also showed an increased rate of NADP+ dissociation, and thus an enhanced substrate reduction with NADPH. Addition of a negatively-charged amino acid residue at the first position afrer the second beta-strand (Tyr49Asp) affected the coenzyme specificity and turned the enzyme into an NAD+-dependent oxidase resembling the cD1d subfamily members.
Descriptors		ASCOMYCETES 17-HYDROXYSTEROID DEHYDROGENASES COENZYMES BINDING SITES MUTAGENESIS, SITE-DIRECTED AMINO ACID SEQUENCE NADP NADPH DEHYDROGENASE