Caractérisation cristallographique d'intermédiaires réactionnels de méthionine sulfoxyde réductases en vue de la compréhension de leur mécanisme catalytique : Les trois domaines de la protéine multifonctionnelle PilB de Neisseria meningitidis et la MsrB de Xanthomonas campestris

Abstract : Methionine residues are easily oxidized to sulfoxides, in vivo. This oxidation is reversed via a ubiquitous enzyme named methionine sulfoxide reductase (Msr). Due to the two possible configurations of the sulfoxide group, two structurally-different classes of enzymes exist: MsrAs are specific for the isomer S, and MsrBs for the isomer R. Both classes act through a two-step mechanism. The first step is dedicated to substrate reduction. It results in the sulfenic form of the catalytic cysteine. Then recycling of the enzyme starts with the formation of an intramolecular disulfide bridge, finally reduced by thioredoxin (Trx). In Neisseria meningitidis, the protein PilB bears MsrA and MsrB as two adjacent domains. A third domain with a Trx-like activity exists at the N-terminal end. The three isolated domains have been studied by X-Ray crystallography. (i) The N-terminal domain: its structure confirmed its homology to Trx and DsbEs, but its analysis revealed elements that probably explain its peculiar properties. (ii) MsrA: the structures of two mutants allowed the observation of a complex with the substrate and of the sulfenic acid. These results add to the structure of the reduced and oxidized forms of the wild type domain so that the catalytic mechanism can be analyzed. (iii) MsrB: the structures of the reduced and oxidized forms were completed by that of a complex with the substrate obtained from a mutant. In addition, comparison with the Xanthomonas campestris MsrB structure enlightened conformational differences between MsrBs from distinct organisms. Finally, structural studies of the whole PilB protein have been initiated in solution using small angle X-Ray scattering.
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Fanomezana Ranaivoson. Caractérisation cristallographique d'intermédiaires réactionnels de méthionine sulfoxyde réductases en vue de la compréhension de leur mécanisme catalytique : Les trois domaines de la protéine multifonctionnelle PilB de Neisseria meningitidis et la MsrB de Xanthomonas campestris. Sciences agricoles. Université Henri Poincaré - Nancy 1, 2007. Français. ⟨NNT : 2007NAN10116⟩. ⟨tel-01748237⟩

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