Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor

摘要

A 1.9 Angstrom resolution X-ray structure of the ape-form of Corynebacterium 2,5-diketo-D-gluconic acid reductase A (2,5-DKGR A), a member of the aldo-keto reductase superfamily, has been determined by molecular replacement using the NADPH-bound form of the same enzyme as the search model. 2,5-DKGR A catalyzes the NADPH-dependent stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate, a precursor in the industrial production of vitamin C. An atomic-resolution structure for the ape-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holoenzyme and hole/substrate model, allows a comparative analysis of structural changes that accompany cofactor binding. The results show that regions of the active site undergo coordinated conformational changes of up to 8 Angstrom. These conformational changes result in the organization and structural rearrangement of residues associated with substrate binding and catalysis. Thus, NADPH functions not only to provide a hydride ion for catalytic reduction, but is also a critical structural component for formation of a catalytically competent form of DKGR A. (C) 2001 Academic Press. [References: 36]