Investigation of the role of the amino acid residue at position 230 for catalysis in monomeric carbonyl reductase 3.
Monomeric carbonyl reductase 3 (CBR3) is a member of the short-chain dehydrogenase/reductase family. CBR3 exhibits much lower activity than monomeric carbonyl reductase 1 (CBR1) in humans and Chinese hamsters although they are highly homologous to each other in amino acid sequence levels. In the present study, we first cloned the CBR3 gene of rat origin (rCBR3), and characterized its enzymatic activity. rCBR3 also exhibited a limited catalytic efficiency similarly to the other CBR3 orthologues of humans and Chinese hamsters. Among the CBR3 orthologues, the human enzyme showed considerably lower activity. Compared with the amino acid sequences of CBR1 and CBR3 among humans, rats, Chinese hamsters, and mice, the tryptophan residue at position 230 is highly conserved while human CBR3 possesses rigid amino acid, proline, at that position instead. Thus, the Trp-230 was expected to be one of the important residues for catalysis since it locates in the hinge region at the substrate-binding loop. The substitution of tryptophan for proline in hCBR3 failed to affect the enzymatic characteristics. Similarly, the substitution of proline for tryptophan in either Chinese hamster CBR3 (CHCR3) or rCBR3 showed no significant change in the catalytic properties. These results suggest that limited catalytic efficiency of carbonyl reductase activity of CBR3 is a common property among animal species, and the substitution of the amino acid residue at position 230 alone has no apparent impact on their enzymatic activities.
Miura
Takeshi
T
Faculty of Pharmacy, Laboratory of Biochemistry, Osaka Ohtani University, Tondabayashi, Osaka, Japan. miurat@osaka-ohtani.ac.jp
Itoh
Yuma
Y
Takada
Masahito
M
Tsutsui
Hidenobu
H
Yukimura
Tokihito
T
Nishinaka
Toru
T
Terada
Tomoyuki
T
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)