MOLECULAR-CLONING, EXPRESSION AND CATALYTIC ACTIVITY OF A HUMAN AKR7 MEMBER OF THE ALDO-KETO REDUCTASE SUPERFAMILY - EVIDENCE THAT THE MAJOR 2-CARBOXYBENZALDEHYDE REDUCTASE FROM HUMAN LIVER IS A HOMOLOG OF RATAFLATOXIN B-1 ALDEHYDE REDUCTASE

The masking of charged amino or carboxy groups by N-phthalidylation an d O-phthalidylation has been used to improve the absorption of many dr ugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxy benzaldehyde (2-CBA) and the pharmaceutically active compound; in huma ns, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by red uction of the aldehyde group. In the present work, the enzyme responsi ble for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B-1-aldehyde reductase (rAFAR). This novel human aldo -keto reductase (AKR) has been cloned from a liver cDNA library, and t ogether with the rat protein, establishes the AKR7 family of the AKR s uperfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in e xtrahepatic tissues. The deduced human and rat protein sequences share 78 % identity and 87 % similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which dis tinguish them from the prototypic AKR1 family of AKRs, the catalytic-a nd NADPH-binding residues appear to be conserved in both families. Cer tain of the predicted structural features of the AKR7 family members a re shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B-1-8,9-dihydr odiol, hAFAR shows high affinity for the gamma-aminobutyric acid metab olite succinic semialdehyde (SSA) which is structurally related to 2-C BA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein.