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Theses

Exploration expérimentale du métabolisme du soufre dans le vivant

Abstract : Acinetobacter baylyi ADP1 (ADP1), a soil bacterium with remarkable aromatic molecule degradation capacities is used in the laboratory as a model organism for the elucidation of new enzymatic functions and metabolic pathways. Taking advantage of two qualities of ADP1 (natural competence and effective homologous recombination), a complete collection of knock-out mutants has been obtained. Analysis of this genomic resource revealed unpredicted phenotypes by the annotation, leading in particular to the initiation of the study of sulphur metabolism in ADP1 with two axes: (1) the biosynthesis of L-methionine (L-Met) and more specifically the study of the families of non-homologous MetX and MetA enzymes catalysing the acylation of homoserine, the first step of this pathway; (2) the recycling of L-methionine. The thesis falls within these two themes. First, a vast project combining experimental screening and structural analysis of the active sites of MetA and MetX had previously made it possible to identify the residues determining the use of acyl-CoA and to propose precise function prediction rules for these two finally isofunctional families. Among other things, this study revealed that 10% of MetX were not involved in L-Met biosynthesis. We have therefore undertaken the characterization of these paralogues with novel activity L-serine O-succinyltransferase (SST) and finally involved in the biosynthesis of L-cysteine (L-Cys). Until then, acetylation by L-serine O-acetyltransferases (SAT) was the only known way to activate L-serine. The determination of kinetic parameters of SST as well as in vitro LCMS characterization and in vivo detection of O-succinyl-L-serine (OSS) in the metabolomes of Schizosaccharomyces pombe yeast and Xanthomonas campestris bacterium demonstrated the function of these paralogues. To complete the demonstration, the characterization of the respective cysteine synthases (CysK) showed that they indeed perform sulfhydrylation of OSS to form L-Cys. Our study also revealed that the described pathway of L-cysteine biosynthesis in yeasts hitherto extrapolated from the model yeast Saccharomyces cerevisae (reverse transsulfuration pathway) was actually an exception and that the vast majority of yeasts synthesize L-Cys from L-serine via this new metabolite, OSS. This thesis, in a second stage, initiated the experimental exploration of sulphur assimilation pathways in ADP1, in which no L-Met recycling pathway was predicted even though it is a source of sulphur. Combining complementary approaches (reverse genetics by phenotyping the complete collection of mutants on various sulphur sources, transcriptomics on L-Met and L-Cys versus sulphate, screening of ADP1 PLP enzymes, biochemical study of ADP1 candidates and mutants), a fairly accurate picture of the assimilation pathways of various sulphur sources is emerging. For example, the assimilation pathways of L-Met and DMSP appear to lead to the production of sulphite via methanesulphonate synthesis under the control of the transcriptional regulator CBL. In addition, KMBA and methanethiol are probably intermediates in the L-Met recycling pathway, whereas DMSO and DMSO2 appear to be catabolic intermediates only for DMSP.
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  • HAL Id : tel-03329174, version 1

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Thomas Bessonnet. Exploration expérimentale du métabolisme du soufre dans le vivant. Médecine humaine et pathologie. Université Paris Saclay (COmUE), 2018. Français. ⟨NNT : 2018SACLE035⟩. ⟨tel-03329174⟩

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