Structural basis for substrate specificity in human monomeric carbonyl reductases.
Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3.


BACKGROUND
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Structural Genomics Consortium, University of Oxford, Headington, United Kingdom.
Ewa S
ES
Pilka
Frank H
FH
Niesen
Wen Hwa
WH
Lee
Yasser
Y
El-Hawari
James E
JE
Dunford
Grazyna
G
Kochan
Vladimir
V
Wsol
Hans-Joerg
HJ
Martin
Edmund
E
Maser
Udo
U
Oppermann
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000970:Antineoplastic Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D004983:Ethanolamines / Q000737:chemistry; D006801:Humans; D007546:Isoquinolines / Q000737:chemistry; D007700:Kinetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D015262:Xenobiotics / Q000737:chemistry
D000970:Antineoplastic Agents; D004983:Ethanolamines; D007546:Isoquinolines; D015262:Xenobiotics; C103439:oracine; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; C531129:CBR1 protein, human; C531130:CBR3 protein, human; D000449:Aldehyde Reductase