Abstract
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.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Highlights
Reduction of carbonyl groups to the corresponding alcohols constitutes a significant metabolic step both for endogenous and xenobiotic compounds [1,2,3]
A preference of carbonyl reductase 3 (CBR3) for orthoquinones is apparent; no activity was found towards menadione, one of the standard substrates used in activity screens for carbonyl reductases
This work establishes the structural basis for narrower substrate specificity in CBR3, and highlights the active site loop found in CBRs as flexible entity that is one critical factor for substrate specificity
Summary
Reduction of carbonyl groups to the corresponding alcohols constitutes a significant metabolic step both for endogenous and xenobiotic compounds [1,2,3]. These reactions are carried out by distinct NAD(P)(H) dependent oxidoreductases mainly belonging to three protein superfamilies, namely the short-chain dehydrogenases/reductases (SDR), aldo-keto-reductases (AKR), or mediumchain dehydrogenases/reductases (MDR) [1,2,3]. The major cytosolic enzymes identified are the NADPH-dependent carbonyl reductase (CBR1, according to the official nomenclature system SDR21C1)[4,5], belonging to the SDR family, and members of the AKR family such as aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), several dihydrodiol/hydroxysteroid dehydrogenases (of the AKR1C subfamily) or aflatoxin aldehyde reductase (AKR7A2) [1,2,3,6]
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