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Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.

ABSTRACT: 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.

SUBMITTER: Poelarends GJ

PROVIDER: S-EPMC2596063 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.

Poelarends Gerrit J GJ Almrud Jeffrey J JJ Serrano Hector H Darty Joseph E JE Johnson William H WH Hackert Marvin L ML Whitman Christian P CP

Biochemistry 20060601 25

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, cat ...[more]

PMID: 16784221

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Project description:The tautomerase superfamily consists of structurally homologous proteins that are characterized by a ?-?-? fold and a catalytic amino-terminal proline. 4-Oxalocrotonate tautomerase (4-OT) family members have been identified and categorized into five subfamilies on the basis of multiple sequence alignments and the conservation of key catalytic and structural residues. Representative members from two subfamilies have been cloned, expressed, purified, and subjected to kinetic and structural characterization. The crystal structure of DmpI from Helicobacter pylori (HpDmpI), a 4-OT homolog in subfamily 3, has been determined to high resolution (1.8Å and 2.1Å) in two different space groups. HpDmpI is a homohexamer with an active site cavity that includes Pro-1, but lacks the equivalent of Arg-11 and Arg-39 found in 4-OT. Instead, the side chain of Lys-36 replaces that of Arg-11 in a manner similar to that observed in the trimeric macrophage migration inhibitory factor (MIF), which is the title protein of another family in the superfamily. The electrostatic surface of the active site is also quite different and suggests that HpDmpI might prefer small, monoacid substrates. A kinetic analysis of the enzyme is consistent with the structural analysis, but a biological role for the enzyme remains elusive. The crystal structure of DmpI from Archaeoglobus fulgidus (AfDmpI), a 4-OT homolog in subfamily-4, has been determined to 2.4Å resolution. AfDmpI is also a homohexamer, with a proposed active site cavity that includes Pro-1, but lacks any other residues that are readily identified as catalytic ones related to 4-OT activity. Indeed, the electrostatic potential of the active site differs significantly in that it is mostly neutral, in contrast to the usual electropositive features found in other 4-OT family members, suggesting that AfDmpI might accommodate hydrophobic substrates. A kinetic analysis has been carried out, but does not provide any clues about the type of reaction the enzyme might catalyze.

Dataset Information

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.

Publications

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.

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