Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding.
OBJECTIVE
Monomeric carbonyl reductase 1 (CBR1) and 3 (CBR3) are members of the short-chain dehydrogenase/reductase superfamily, and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR3 exhibits a higher K(m) value toward NADPH and more limited carbonyl reductase activities than CBR1, although they are highly homologous to each other in amino acid sequence levels. In the present study, we investigated the origin of the different properties of the enzymes by analyses using several chimeric enzymes.


METHODS
Harr-plot analysis of the amino acid sequences was conducted and as a result, two low-identity regions between human CBR1 and CBR3 were found: these were designated as the N-terminal low-identity region (LirN) and the C-terminal low-identity region (LirC; the substrate-binding region). We genetically constructed chimeric enzymes while focusing on these regions.


RESULTS
Chimeric CBR1 possessing LirN of CBR3 (CBR1LirN3) exhibited CBR1-like activities but a low coenzyme affinity probably due to a structural alteration in a micro domain, whereas chimeric CBR1 including LirC of CBR3 (CBR1LirC3) was enzymatically similar to CBR3. Furthermore, CBR3LirC1 was similar to CBR1 in both enzymatic activities and coenzyme binding.


CONCLUSIONS
These results suggested that LirC, i.e., the substrate-binding loop region, is the origin of the difference between human CBR1 and CBR3 in both catalytic and coenzyme-binding properties.
Miura
Takeshi
T
Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan. miurat@osaka-ohtani.ac.jp
Nishinaka
Toru
T
Terada
Tomoyuki
T
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism*; D019295:Computational Biology; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D003067:Coenzymes; D000429:Alcohol Oxidoreductases; C531129:CBR1 protein, human; C531130:CBR3 protein, human