Dataset Information

Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1.

ABSTRACT: Methylibium petroleiphilum strain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized. Tautomerase I and tautomerase II have the lowest pairwise sequence identity (35%) among the isozyme pairs in the parallel pathways, and could offer insight into substrate preferences and pathway functions. The kinetic parameters of tautomerase I and tautomerase II were determined using 2-hydroxymuconate and 5-(methyl)-2-hydroxymuconate. Both tautomerase I and tautomerase II process the substrates, but with different efficiencies. Crystal structures were determined for both tautomerase I and tautomerase II, at 1.57 and 1.64Å resolution, respectively. The backbones of tautomerase I and tautomerase II are highly similar, but have distinct active site environments. The results, in combination with those for other structurally and kinetically characterized 4-OT isozymes, suggest that tautomerase I catalyzes the tautomerization of both 2-hydroxymuconate and alkyl derivatives, whereas tautomerase II might specialize in other aromatic hydrocarbon metabolites.

SUBMITTER: Terrell CR

PROVIDER: S-EPMC3788618 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1.

Terrell Cassidy R CR Burks Elizabeth A EA Whitman Christian P CP Hoffman David W DW

Archives of biochemistry and biophysics 20130704 1

Methylibium petroleiphilum strain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized. Tautomerase I and tautomerase II have the lowest pairwis ...[more]

PMID: 23831510

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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

Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1.

Publications

Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1.

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