Site-Directed Mutagenesis of Residues in Coenzyme-Binding Domain and Active Site of Mouse Lung Carbonyl Reductase

Abstract

Tetrameric carbonyl reductase (CR) [EC 1.1.1.184] in mouse, guinea-pig and pig lungs catalyzes the reduction of various carbonyl compounds and the oxidation of secondary alcohols with NADP(H) as preferable coenzymes to NAD(H) (Nakayama, 1986; Matsuura, 1988; Oritani, 1992). The cDNAs for pulmonary CRs of pig and mouse have been cloned (Nakanishi, 1993; 1995), and the cDNA for mouse lung CR is identical with that for an mRNA which is increased in differentiation of murine adipocytes (Navre and Ringold, 1988). Their deduced amino acid sequences, composed of 244 residues (85% identity between them), are structurally related to members of the short-chain dehydrogenase/reductase (SDR) family, which includes a large number of prokaryotic and eukaryotic enzymes with different specificities for coenzymes and substrates (Jornvall, 1995). The pulmonary CRs conserve two sequences, Gly-X-X-X-Gly-X-Gly and Tyr-X-X-X-Lys, which have been demonstrated to be in the coenzyme-binding domain and the active site, respectively, by site-directed mutagenesis (Jornvall, 1995; and references cited therein) and X-ray crystallographic studies (Varughese, 1994; Ghosh, 1994; 1995; Tanaka, 1996b) of several NAD(H)-dependent SDR family proteins. The region around the former sequence forms a βαβ-fold which is characteristic of the coenzyme binding fold in dehydrogenases of other families (Wierenga, 1985;1986). The Tyr residue in the latter sequence plays the most essential role as the catalyst in the reaction mechanism, and the side-chain of the Lys residue stabilizes the ionized form of the Tyr.