Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+.
Aldo-keto reductases of family 2 employ single site replacement Lys-->Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3A, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.
Leitgeb
Stefan
S
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Petschacher
Barbara
B
Wilson
David K
DK
Nidetzky
Bernd
B
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D001120:Arginine / Q000378:metabolism*; D002175:Candida / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation
D003067:Coenzymes; D009243:NAD; D009249:NADP; D001120:Arginine; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D008239:Lysine