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| | {{STRUCTURE_2fm7| PDB=2fm7 | SCENE= }} | | {{STRUCTURE_2fm7| PDB=2fm7 | SCENE= }} |
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| - | '''Evolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase'''
| + | ===Evolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase=== |
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| - | ==Overview==
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| - | 4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_16784221}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 16784221 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_16784221}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Mutant]] | | [[Category: Mutant]] |
| | [[Category: Tautomerase]] | | [[Category: Tautomerase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 04:03:34 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 01:05:13 2008'' |
Revision as of 22:05, 28 July 2008
Template:STRUCTURE 2fm7
Evolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase
Template:ABSTRACT PUBMED 16784221
About this Structure
2FM7 is a Single protein structure of sequence from Pseudomonas putida. Full crystallographic information is available from OCA.
Reference
Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase., Poelarends GJ, Almrud JJ, Serrano H, Darty JE, Johnson WH Jr, Hackert ML, Whitman CP, Biochemistry. 2006 Jun 27;45(25):7700-8. PMID:16784221
Page seeded by OCA on Tue Jul 29 01:05:13 2008
