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| - | [[Image:1sm9.jpg|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1sm9| PDB=1sm9 | SCENE= }} | | {{STRUCTURE_1sm9| PDB=1sm9 | SCENE= }} |
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| - | '''Crystal Structure Of An Engineered K274RN276D Double Mutant of Xylose Reductase From Candida Tenuis Optimized To Utilize NAD'''
| + | ===Crystal Structure Of An Engineered K274RN276D Double Mutant of Xylose Reductase From Candida Tenuis Optimized To Utilize NAD=== |
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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_15320875}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15320875 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_15320875}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Coenzyme specificity]] | | [[Category: Coenzyme specificity]] |
| | [[Category: Xylose metabolism]] | | [[Category: Xylose metabolism]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 08:52:43 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 02:44:14 2008'' |
Revision as of 23:44, 27 July 2008
Template:STRUCTURE 1sm9
Crystal Structure Of An Engineered K274RN276D Double Mutant of Xylose Reductase From Candida Tenuis Optimized To Utilize NAD
Template:ABSTRACT PUBMED 15320875
About this Structure
1SM9 is a Single protein structure of sequence from Candida tenuis. 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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