The Role of Cys-298 in Aldose Reductase Function

Ganesaratnam K Balendiran,Michael R Sawaya,Frederick P Schwarz,Gomathinayagam Ponniah,Richard Cuckovich,Malkhey Verma,Duilio Cascio

doi:10.1074/jbc.m110.154195

Abstract

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

Highlights

The aldo-keto reductases constitute a superfamily of NADPH-dependent oxidoreductases [1] that catalyze the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols
Of the six cysteines found in Human AR (hAR), only Cys298 is located in sufficient proximity to the active site to directly perturb both enzyme activity and inhibitor sensitivity
Production of Recombinant Wild-type and C298S hAR— His-tagged recombinant wild-type and C298S mutant hAR were expressed in Escherichia coli BL21 cells that were grown in Luria-Bertani broth containing 50 mg/liter ampicillin with constant shaking in rotary shaker at 240 rpm to reach the A600 between 0.6 and 0.8 at 37 °C

Summary

EXPERIMENTAL PROCEDURES

Production of Recombinant Wild-type and C298S hAR— His-tagged recombinant wild-type and C298S mutant hAR were expressed in Escherichia coli BL21 cells that were grown in Luria-Bertani broth containing 50 mg/liter ampicillin with constant shaking in rotary shaker at 240 rpm to reach the A600 between 0.6 and 0.8 at 37 °C. After initial rigidbody and simulated annealing refinements, electron density maps with 2 Fo Ϫ Fc and Fo Ϫ Fc coefficients were employed to rebuild the mutant protein structure and to locate the cofactor and solvent molecules following the procedures described above. Both the final models were validated with the following structure validation tools: PROCHECK [33], ERRAT [34], and VERIFY3D [35].

RESULTS

Diffraction data

DISCUSSION

NADP atoms