1SM9

Crystal Structure Of An Engineered K274RN276D Double Mutant of Xylose Reductase From Candida Tenuis Optimized To Utilize NAD

Summary for 1SM9

• Entry DOI: 10.2210/pdb1sm9/pdb
• Descriptor: xylose reductase, NICOTINAMIDE-ADENINE-DINUCLEOTIDE (3 entities in total)
• Functional Keywords: xylose metabolism, coenzyme specificity, aldo-keto reductase, beta-alpha-barrel, akr2b5, oxidoreductase
• Biological source: Candida tenuis
• Total number of polymer chains: 4
• Total formula weight: 147018.50

Authors

Petschacher, B.,Leitgeb, S.,Kavanagh, K.L.,Wilson, D.K.,Nidetzky, B. (deposition date: 2004-03-08, release date: 2004-12-21, Last modification date: 2023-08-23)

Primary citation

Petschacher, B.,Leitgeb, S.,Kavanagh, K.L.,Wilson, D.K.,Nidetzky, B.
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.
Biochem.J., 385:75-83, 2005

PubMed Abstract: 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.

PubMed: 15320875
DOI: 10.1042/BJ20040363

Experimental method

X-RAY DIFFRACTION (2.2 Å)