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Structural and functional characterisation of the methionine adenosyltransferase from Thermococcus kodakarensis.

ABSTRACT: BACKGROUND: Methionine adenosyltransferases catalyse the synthesis of S-adenosylmethionine, a cofactor abundant in all domains of life. In contrast to the enzymes from bacteria and eukarya that show high sequence similarity, methionine adenosyltransferases from archaea diverge on the amino acid sequence level and only few conserved residues are retained. RESULTS: We describe the initial characterisation and the crystal structure of the methionine adenosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis. As described for other archaeal methionine adenosyltransferases the enzyme is a dimer in solution and shows high temperature stability. The overall structure is very similar to that of the bacterial and eukaryotic enzymes described, with some additional features that might add to the stability of the enzyme. Compared to bacterial and eukaryotic structures, the active site architecture is largely conserved, with some variation in the substrate/product-binding residues. A flexible loop that was not fully ordered in previous structures without ligands in the active side is clearly visible and forms a helix that leaves an entrance to the active site open. CONCLUSIONS: The similar three-dimensional structures of archaeal and bacterial or eukaryotic methionine adenosyltransferases support that these enzymes share an early common ancestor from which they evolved independently, explaining the low similarity in their amino acid sequences. Furthermore, methionine adenosyltransferase from T. kodakarensis is the first structure without any ligands bound in the active site where the flexible loop covering the entrance to the active site is fully ordered, supporting a mechanism postulated earlier for the methionine adenosyltransferase from E. coli. The structure will serve as a starting point for further mechanistic studies and permit the generation of enzyme variants with different characteristics by rational design.

SUBMITTER: Schlesier J

PROVIDER: S-EPMC3853416 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Structural and functional characterisation of the methionine adenosyltransferase from Thermococcus kodakarensis.

Schlesier Julia J Siegrist Jutta J Gerhardt Stefan S Erb Annette A Blaesi Simone S Richter Michael M Einsle Oliver O Andexer Jennifer N JN

BMC structural biology 20131018

<h4>Background</h4>Methionine adenosyltransferases catalyse the synthesis of S-adenosylmethionine, a cofactor abundant in all domains of life. In contrast to the enzymes from bacteria and eukarya that show high sequence similarity, methionine adenosyltransferases from archaea diverge on the amino acid sequence level and only few conserved residues are retained.<h4>Results</h4>We describe the initial characterisation and the crystal structure of the methionine adenosyltransferase from the hyperth ...[more]

PMID: 24134203

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Project description:BackgroundTuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.ResultsThe gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM.ConclusionThe methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.

Project description:Members of Thermococcales harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P csg ). The purified protein did not display aminotransferase activity but exhibited racemase activity. An examination of most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal medium supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase but displayed cell yield similar to that observed in medium with l-Leu. In contrast, the ΔTK1211 strain displayed growth in medium with l-Leu but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensisIMPORTANCE Phylogenetic analysis of aminotransferase class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups now can be presumed to function as a broad-substrate-specificity amino acid racemase (subgroup 1), alanine/serine racemase (subgroup 2), ornithine ω-aminotransferase (subgroup 3), or Leu/Met racemase (subgroup 4).

Dataset Information

Structural and functional characterisation of the methionine adenosyltransferase from Thermococcus kodakarensis.

Publications

Structural and functional characterisation of the methionine adenosyltransferase from Thermococcus kodakarensis.

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