A member of the aldo-keto reductase (AKR) protein superfamily, AKR1B10, is overexpressed in human liver cancers as well as in many adenocarcinoma cases due to smoking. AKR1B10 is also detected in instances of cervical and endometrial cancer in uterine cancer patients. In addition, AKR1B10 has been identifiedasabiomarkerfornon-small-celllungcancerbyacombinedbioinformaticsandclinicalanalysis. Furthermore, in breast cancer cells, fatty acid biosynthesis is regulated by AKR1B10. AKR1B10 contains 316 residues, shares 70% sequence identity with aldose reductase (AKR1B1) and has the conserved Cys residue at position 299. Carbonyl groups in some anticancer drugs and dl-glyceraldehyde are converted by AKR1B10 to their corresponding alcohols. The anticancer drug daunorubicin, which is currently used in the clinical treatment of various forms of cancer, is converted by AKR1B10 to daunorubicinol with a Km and kcat of 1.1±0.18 mM and 1.4±0.16min−1, respectively. This carbonyl reducing activity of AKR1B10 decreases the anticancer effectiveness of daunorubicin. Similarly, kinetic parameters Km and kcat (NADPH, DL-glyceraldehyde) for the reduction of dl-glyceraldehyde by wild-type AKR1B10 are 2.2±0.2mM and 0.71±0.05sec−1, respectively. Mutation of residue 299 from Cys to Ser in AKR1B10 reduces the protein affinity for dl-glyceraldehyde and enhances AKR1B10’s catalytic activity but overall catalytic efficiency is reduced. For dl-glyceraldehyde reduction that is catalyzed by the Cys299Ser mutant AKR1B10, Km is 15.8±1.0mM and kcat (NADPH, DL-glyceraldehyde) is 2.8±0.2sec−1. This implies that the substrate specificity of AKR1B10 is drastically affected by mutation of residue 299 from Cys to Ser. In the present paper, we use this mutation in AKR1B10 to characterize a library of compounds regarding their different inhibitory potency on the carbonyl reducing activity of wild-type and the Cys299Ser mutant AKR1B10.

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.

Carbonyl reduction is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics. Carbonyl reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, has been poorly characterized so far, and the regulation of its expression is a complete mystery. Here, we show that CBR3 expression is regulated via Nrf2, a key regulator in response to oxidative stress. In human cancer cell lines, CBR3 mRNA was expressed differentially, ranging from very high (A549, lung) to very low (HT-29, colon; HepG2, liver) levels. CBR3 protein was highly expressed in SW-480 (colon) cells but was absent in HCT116 (colon) and HepG2 cells. CBR3 mRNA could be induced in HT-29 cells by Nrf2 agonists [sulforaphane (SUL, 7-fold) and diethyl maleate (DEM, 4-fold)] or hormone receptor ligand Z-guggulsterone (5-fold). Aryl hydrocarbon receptor agonist B[k]F failed to induce CBR3 mRNA after incubation for 8 h but elevated CBR3 levels after 24 h, most likely mediated by B[k]F metabolites that can activate Nrf2 signaling. Inhibition of Nrf2-activating upstream kinase MEK/ERK by PD98059 weakened DEM-mediated induction of CBR3 mRNA. Proteasome inhibitors MG-132 (5 μM) and bortezomib (50 nM) dramatically increased the level of CBR3 mRNA, obviously because of the increase in the level of Nrf2 protein. While siRNA-mediated knockdown of Nrf2 led to a decrease in the level of CBR3 mRNA in A549 cells (30% of control), Keap1 knockdown increased the level of CBR3 mRNA expression in HepG2 (9.3-fold) and HT-29 (2.7-fold) cells. Here, we provide for the first time evidence that human CBR3 is a new member of the Nrf2 gene battery.

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