1hqt

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(New page: 200px<br /><applet load="1hqt" size="450" color="white" frame="true" align="right" spinBox="true" caption="1hqt, resolution 2.2&Aring;" /> '''THE CRYSTAL STRUCTURE...)
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'''THE CRYSTAL STRUCTURE OF AN ALDEHYDE REDUCTASE Y50F MUTANT-NADP COMPLEX AND ITS IMPLICATIONS FOR SUBSTRATE BINDING'''<br />
'''THE CRYSTAL STRUCTURE OF AN ALDEHYDE REDUCTASE Y50F MUTANT-NADP COMPLEX AND ITS IMPLICATIONS FOR SUBSTRATE BINDING'''<br />
==Overview==
==Overview==
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In order to understand more fully the structural features of aldo-keto, reductases (AKRs) that determine their substrate specificities it would be, desirable to obtain crystal structures of an AKR with a substrate at the, active site. Unfortunately the reaction mechanism does not allow a binary, complex between enzyme and substrate and to date ternary complexes of, enzyme, NADP(H) and substrate or product have not been achieved. Previous, crystal structures, in conjunction with numerous kinetic and theoretical, analyses, have led to the general acceptance of the active site tyrosine, as the general acid-base catalytic residue in the enzyme. This view is, supported by the generation of an enzymatically inactive site-directed, mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]., However, crystallization of this mutant was unsuccessful. We have, attempted to generate a trapped cofactor/substrate complex in pig aldehyde, reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed, mutant. We have been successful in the generation of the first high, resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable, to generate any ternary complexes. The binary complex was refined to 2.2A, and shows a clear lack of density due to the missing hydroxyl group. Other, residues in the active site are not significantly perturbed when compared, to other available reductase structures. The mutant binds cofactor (both, oxidized and reduced) more tightly but shows a complete lack of binding of, the aldehyde reductase inhibitor barbitone as determined by fluorescence, titrations. Attempts at substrate addition to the active site, either by, cocrystallization or by soaking, were all unsuccessful using, pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex, formation, combined with the significant differences in the binding of, barbitone provides some experimental proof of the proposal that the, hydroxyl group on the active site tyrosine is essential for substrate, binding in addition to its major role in catalysis. We propose that the, initial event in catalysis is the binding of the oxygen moiety of the, carbonyl-group of the substrate through hydrogen bonding to the tyrosine, hydroxyl group.
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In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.
==About this Structure==
==About this Structure==
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1HQT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Sus_scrofa Sus scrofa] with NAP as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Alcohol_dehydrogenase_(NADP(+)) Alcohol dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.2 1.1.1.2] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1HQT OCA].
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1HQT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Sus_scrofa Sus scrofa] with <scene name='pdbligand=NAP:'>NAP</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Alcohol_dehydrogenase_(NADP(+)) Alcohol dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.2 1.1.1.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1HQT OCA].
==Reference==
==Reference==
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[[Category: Single protein]]
[[Category: Single protein]]
[[Category: Sus scrofa]]
[[Category: Sus scrofa]]
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[[Category: Flynn, T.G.]]
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[[Category: Flynn, T G.]]
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[[Category: Green, N.C.]]
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[[Category: Green, N C.]]
[[Category: Hyndman, D.]]
[[Category: Hyndman, D.]]
[[Category: Jia, Z.]]
[[Category: Jia, Z.]]
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[[Category: alpha/beta barrel tim barrel holo enzyme]]
[[Category: alpha/beta barrel tim barrel holo enzyme]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 16:45:36 2007''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:03:56 2008''

Revision as of 11:03, 21 February 2008


1hqt, resolution 2.2Å

Drag the structure with the mouse to rotate

THE CRYSTAL STRUCTURE OF AN ALDEHYDE REDUCTASE Y50F MUTANT-NADP COMPLEX AND ITS IMPLICATIONS FOR SUBSTRATE BINDING

Overview

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

About this Structure

1HQT is a Single protein structure of sequence from Sus scrofa with as ligand. Active as Alcohol dehydrogenase (NADP(+)), with EC number 1.1.1.2 Full crystallographic information is available from OCA.

Reference

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding., Ye Q, Hyndman D, Green NC, Li L, Jia Z, Flynn TG, Chem Biol Interact. 2001 Jan 30;130-132(1-3):651-8. PMID:11306083

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