Studies on the inhibitor-binding site of porcine aldehyde reductase: Crystal structure of the holoenzyme-inhibitor ternary complex

Abstract

Aldehyde reductase is an enzyme capable of metabolizing a wide variety of aldehydes to their corresponding alcohols. The tertiary structures of aldehyde reductase and aldose reductase are similar and consist of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. We have determined the X-ray crystal structure of porcine aldehyde reductase holoenzyme in complex with an aldose reductase inhibitor, tolrestat, at 2.4 A resolution to obtain a picture of the binding conformation of inhibitors to aldehyde reductase. Tolrestat binds in the active site pocket of aldehyde reductase and interacts through van der Waals contacts with Arg 312 and Asp 313. The carboxylate group of tolrestat is within hydrogen bonding distance with His 113 and Trp 114. Mutation of Arg 312 to alanine in porcine aldehyde reductase alters the potency of inhibition of the enzyme by aldose reductase inhibitors. Our results indicate that the structure of the inhibitor-binding site of aldehyde reductase differs from that of aldose reductase due to the participation of nonconserved residues in its formation. A major difference is the participation of Arg 312 and Asp 313 in lining the inhibitor-binding site in aldehyde reductase but not in aldose reductase.

Disruption of Aldehyde Reductase Increases Group Size in Dictyostelium

“…1). An NCBI protein-protein BLAST search (available on the World Wide Web at www.ncbi.nlm.nih.gov/blast/) using the deduced amino acid sequence of the open reading frame found a 45% identity to porcine alcohol dehydrogenase [NADP ϩ ] (aldehyde reductase) (48,49) and a 44% identity to human (50) and mouse (51) aldehyde reductase and to a putative Arabidopsis mannose-6-phosphate reductase (NADPH-dependent) protein (gi: 15226502). The NCBI conserved domain search performed in conjunction with the BLAST search found the Dictyostelium protein identified above to be 99.6% aligned with the aldo/keto reductase family and 95.7% aligned with the ARA1 aldo/keto reductase-diketogulonate reductase family signatures.…”

Aldose Reductase Inhibitors

“…A significantly different interaction is expressed in the trifluoromethyl group of tolrestat. This group contacts the side chains of Arg312 and Asp313 in ALR; 45 however, it forms hydrogen bonds in AR. Interestingly, the mutation Arg312Ala in ALR induces 130-fold tighter bindings of tolrestat.…”

“…Interestingly, the mutation Arg312Ala in ALR induces 130-fold tighter bindings of tolrestat. 45 The superimposition of AR with zopolrestat and ALR with tolrestat illustrates zopolrestat's differing interactions between these two enzymes. 46 Regarding the hydrophilic interactions of zopolrestat, three residues (Cys298, Leu300, and Thr113) participate in hydrogen bondings with the atoms on the phthalazinone ring and benzothiazole ring in AR, as described in the previous section; however, the corresponding residues (isoleucine, proline, and tyrosine, respectively) in ALR cannot form hydrogen bonds.…”

Crystal structure of the Escherichia coli Tas protein, an NADP(H)‐dependent aldo‐keto reductase

“…All members of the family have a (␤/␣) 8 TIM barrel fold. The catalytic site with the bound NADP(H) cofactor is located at the C-terminal edge of the ␤-strands.…”

“…Loops surrounding the active site form the substrate recognition cylindrical surface. Crystal complexes with various inhibitors [5][6][7][8] have indicated the ability of aldoketo reductases to accommodate a wide range of substrates due to the conformational flexibility of the recognition loops.…”