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Characterization of the Thermotoga maritima chemotaxis methylation system that lacks pentapeptide-dependent methyltransferase CheR:MCP tethering.

ABSTRACT: Sensory adaptation in bacterial chemotaxis is mediated by covalent modifications of specific glutamate and glutamine residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli and Salmonella enterica, efficient methylation of MCPs depends on the localization of methyltransferase CheR to MCP clusters through an interaction between the CheR beta-subdomain and a pentapeptide sequence (NWETF or NWESF) at the C-terminus of the MCP. In vitro methylation analyses utilizing S. enterica and Thermotoga maritima CheR proteins and MCPs indicate that MCP methylation in T. maritima occurs independently of a pentapeptide-binding motif. Kinetic and binding measurements demonstrate that despite efficient methylation, the interaction between T. maritima CheR and T. maritima MCPs is of relatively low affinity. Comparative protein sequence analyses of CheR beta-subdomains from organisms having MCPs that contain and/or lack pentapeptide-binding motifs identified key similarities and differences in residue conservation, suggesting the existence of two distinct classes of CheR proteins: pentapeptide-dependent and pentapeptide-independent methyltransferases. Analysis of MCP C-terminal ends showed that only approximately 10% of MCPs contain a putative C-terminal binding motif, the majority of which are restricted to the different proteobacteria classes (alpha, beta, gamma, delta). These findings suggest that tethering of CheR to MCPs is a relatively recent event in evolution and that the pentapeptide-independent methylation system is more common than the well-characterized pentapeptide-dependent methylation system.

SUBMITTER: Perez E

PROVIDER: S-EPMC3645907 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Characterization of the Thermotoga maritima chemotaxis methylation system that lacks pentapeptide-dependent methyltransferase CheR:MCP tethering.

Perez Eduardo E Stock Ann M AM

Molecular microbiology 20061205 2

Sensory adaptation in bacterial chemotaxis is mediated by covalent modifications of specific glutamate and glutamine residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli and Salmonella enterica, efficient methylation of MCPs depends on the localization of methyltransferase CheR to MCP clusters through an interaction between the CheR beta-subdomain and a pentapeptide sequence (NWETF or NWESF) at the C-terminus of the MCP. In vitro methylation ...[more]

PMID: 17163981

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Project description:Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

Dataset Information

Characterization of the Thermotoga maritima chemotaxis methylation system that lacks pentapeptide-dependent methyltransferase CheR:MCP tethering.

Publications

Characterization of the Thermotoga maritima chemotaxis methylation system that lacks pentapeptide-dependent methyltransferase CheR:MCP tethering.

Similar Datasets

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