Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor.
A 1.9 A resolution X-ray structure of the apo-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 stereo-specific 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 apo-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holo-enzyme and holo/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 A. 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.
Sanli
G
G
Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee, FL 32306-4380, USA.
Blaber
M
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000494:Allosteric Regulation; D000495:Allosteric Site; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001205:Ascorbic Acid / Q000096:biosynthesis; D002384:Catalysis; D003352:Corynebacterium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation
D001051:Apoenzymes; D009249:NADP; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D001205:Ascorbic Acid