1z9a
From Proteopedia
(New page: 200px<br /><applet load="1z9a" size="450" color="white" frame="true" align="right" spinBox="true" caption="1z9a, resolution 2.40Å" /> '''Crystal Structure Of...) |
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| - | [[Image:1z9a.gif|left|200px]]<br /><applet load="1z9a" size=" | + | [[Image:1z9a.gif|left|200px]]<br /><applet load="1z9a" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1z9a, resolution 2.40Å" /> | caption="1z9a, resolution 2.40Å" /> | ||
'''Crystal Structure Of The Asn-309 To Asp Mutant Of Candida Tenuis Xylose Reductase (Akr2B5) Bound To Nad+'''<br /> | '''Crystal Structure Of The Asn-309 To Asp Mutant Of Candida Tenuis Xylose Reductase (Akr2B5) Bound To Nad+'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Little is known about how substrates bind to CtXR (Candida tenuis xylose | + | Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol. |
==About this Structure== | ==About this Structure== | ||
| - | 1Z9A is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Candida_tenuis Candida tenuis] with NAD as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | + | 1Z9A is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Candida_tenuis Candida tenuis] with <scene name='pdbligand=NAD:'>NAD</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1Z9A OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Leitgeb, S.]] | [[Category: Leitgeb, S.]] | ||
[[Category: Nidetzky, B.]] | [[Category: Nidetzky, B.]] | ||
| - | [[Category: Wilson, D | + | [[Category: Wilson, D K.]] |
[[Category: NAD]] | [[Category: NAD]] | ||
[[Category: akr]] | [[Category: akr]] | ||
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[[Category: xylose reductase]] | [[Category: xylose reductase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:13:19 2008'' |
Revision as of 14:13, 21 February 2008
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Crystal Structure Of The Asn-309 To Asp Mutant Of Candida Tenuis Xylose Reductase (Akr2B5) Bound To Nad+
Overview
Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol.
About this Structure
1Z9A is a Single protein structure of sequence from Candida tenuis with as ligand. Full crystallographic information is available from OCA.
Reference
Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis., Kratzer R, Leitgeb S, Wilson DK, Nidetzky B, Biochem J. 2006 Jan 1;393(Pt 1):51-8. PMID:16336198
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