1sm9
From Proteopedia
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Crystal Structure Of An Engineered K274RN276D Double Mutant of Xylose Reductase From Candida Tenuis Optimized To Utilize NAD
Overview
CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.
About this Structure
1SM9 is a Single protein structure of sequence from Candida tenuis with as ligand. Active as Aldehyde reductase, with EC number 1.1.1.21 Full crystallographic information is available from OCA.
Reference
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography., Petschacher B, Leitgeb S, Kavanagh KL, Wilson DK, Nidetzky B, Biochem J. 2005 Jan 1;385(Pt 1):75-83. PMID:15320875
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