1xys
From Proteopedia
(New page: 200px<br /><applet load="1xys" size="450" color="white" frame="true" align="right" spinBox="true" caption="1xys, resolution 2.5Å" /> '''CATALYTIC CORE OF XYL...) |
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| - | [[Image:1xys.jpg|left|200px]]<br /><applet load="1xys" size=" | + | [[Image:1xys.jpg|left|200px]]<br /><applet load="1xys" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1xys, resolution 2.5Å" /> | caption="1xys, resolution 2.5Å" /> | ||
'''CATALYTIC CORE OF XYLANASE A E246C MUTANT'''<br /> | '''CATALYTIC CORE OF XYLANASE A E246C MUTANT'''<br /> | ||
==Overview== | ==Overview== | ||
| - | BACKGROUND: Sequence alignment suggests that xylanases evolved from two | + | BACKGROUND: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS: We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS: The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates. |
==About this Structure== | ==About this Structure== | ||
| - | 1XYS is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Cellvibrio_japonicus Cellvibrio japonicus] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Endo-1,4-beta-xylanase Endo-1,4-beta-xylanase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.8 3.2.1.8] Full crystallographic information is available from [http:// | + | 1XYS is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Cellvibrio_japonicus Cellvibrio japonicus] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Endo-1,4-beta-xylanase Endo-1,4-beta-xylanase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.8 3.2.1.8] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1XYS OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Connerton, I.]] | [[Category: Connerton, I.]] | ||
| - | [[Category: Harris, G | + | [[Category: Harris, G W.]] |
| - | [[Category: Jenkins, J | + | [[Category: Jenkins, J A.]] |
| - | [[Category: Pickersgill, R | + | [[Category: Pickersgill, R W.]] |
[[Category: CA]] | [[Category: CA]] | ||
[[Category: family 10 of glycosyl-hydrolase]] | [[Category: family 10 of glycosyl-hydrolase]] | ||
[[Category: family f xylanase]] | [[Category: family f xylanase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:00:05 2008'' |
Revision as of 14:00, 21 February 2008
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CATALYTIC CORE OF XYLANASE A E246C MUTANT
Overview
BACKGROUND: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS: We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS: The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.
About this Structure
1XYS is a Single protein structure of sequence from Cellvibrio japonicus with as ligand. Active as Endo-1,4-beta-xylanase, with EC number 3.2.1.8 Full crystallographic information is available from OCA.
Reference
Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites., Harris GW, Jenkins JA, Connerton I, Cummings N, Lo Leggio L, Scott M, Hazlewood GP, Laurie JI, Gilbert HJ, Pickersgill RW, Structure. 1994 Nov 15;2(11):1107-16. PMID:7881909
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