2d0d
From Proteopedia
(New page: 200px<br /><applet load="2d0d" size="350" color="white" frame="true" align="right" spinBox="true" caption="2d0d, resolution 1.65Å" /> '''Crystal Structure of...) |
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==Overview== | ==Overview== | ||
| - | The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 | + | The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate the substrate specificity and catalytic efficiency of CumD toward substrates derived from monocyclic aromatic compounds, we constructed the CumD mutants, A129V, I199V, and V227I, as well as four types of double and triple mutants. Toward substrates with smaller side chains (e.g. 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the k(cat)/K(m) values of the single mutants were 4.2-11 fold higher than that of the wild type enzyme and 1.8-4.7 fold higher than that of the meta-cleavage product hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the highest k(cat)/K(m) value for 2-hydroxy-6-oxohepta-2,4-dienoate (6-ethyl-HODA). The crystal structure of the A129V mutant was determined at 1.65 A resolution, enabling location of the Ogamma atom of the Ser103 side chain. A chloride ion was bound to the oxyanion hole of the active site, and mutant enzymes at the residues forming this site were also examined. The k(cat) values of Ser34 mutants were decreased 2.9-65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing the anionic oxygen of the proposed intermediate state (gem-diolate). This is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential "triad", being intact. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Fushinobu, S.]] | [[Category: Fushinobu, S.]] | ||
| - | [[Category: Jun, S | + | [[Category: Jun, S Y.]] |
[[Category: Nojiri, H.]] | [[Category: Nojiri, H.]] | ||
[[Category: Omori, T.]] | [[Category: Omori, T.]] | ||
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[[Category: substrate specificity]] | [[Category: substrate specificity]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:54:07 2008'' |
Revision as of 14:54, 21 February 2008
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Crystal Structure of a Meta-cleavage Product Hydrolase (CumD) A129V Mutant
Overview
The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate the substrate specificity and catalytic efficiency of CumD toward substrates derived from monocyclic aromatic compounds, we constructed the CumD mutants, A129V, I199V, and V227I, as well as four types of double and triple mutants. Toward substrates with smaller side chains (e.g. 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the k(cat)/K(m) values of the single mutants were 4.2-11 fold higher than that of the wild type enzyme and 1.8-4.7 fold higher than that of the meta-cleavage product hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the highest k(cat)/K(m) value for 2-hydroxy-6-oxohepta-2,4-dienoate (6-ethyl-HODA). The crystal structure of the A129V mutant was determined at 1.65 A resolution, enabling location of the Ogamma atom of the Ser103 side chain. A chloride ion was bound to the oxyanion hole of the active site, and mutant enzymes at the residues forming this site were also examined. The k(cat) values of Ser34 mutants were decreased 2.9-65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing the anionic oxygen of the proposed intermediate state (gem-diolate). This is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential "triad", being intact.
About this Structure
2D0D is a Single protein structure of sequence from Pseudomonas fluorescens with and as ligands. Active as 2-hydroxymuconate-semialdehyde hydrolase, with EC number 3.7.1.9 Full crystallographic information is available from OCA.
Reference
Improving the catalytic efficiency of a meta-cleavage product hydrolase (CumD) from Pseudomonas fluorescens IP01., Jun SY, Fushinobu S, Nojiri H, Omori T, Shoun H, Wakagi T, Biochim Biophys Acta. 2006 Jul;1764(7):1159-66. Epub 2006 Jun 7. PMID:16844437
Page seeded by OCA on Thu Feb 21 16:54:07 2008
Categories: 2-hydroxymuconate-semialdehyde hydrolase | Pseudomonas fluorescens | Single protein | Fushinobu, S. | Jun, S Y. | Nojiri, H. | Omori, T. | Shoun, H. | Wakagi, T. | CL | PO4 | Alpha/beta-hydrolase | Beta-ketolase | Cumene degradation | Meta-cleavage product hydrolase | Pcb | Polychlorinated biphenyl degradation | Substrate specificity
