Biosynthesis of sulfur-containing compounds – BIOSUF

Sulfur-containing molecules are present in all living organisms where they play essential metabolic roles. They can be divided into inorganic complexes, including iron-sulfur clusters ([Fe-S]), and sulfurated organic compounds such as biotin or lipoic acid. Biosynthesis of sulfur-containing molecules depends upon complex protein machineries and upon a variety of difficult chemical reactions incompletely studied so far. The objective of the project is to understand, by combining the methods of microbial genetics, protein chemistry and bioinformatics, the mechanistic, physiological and evolutionary features of these biosynthetic pathways. This knowledge may have some impact on specific health problems (bacterial infection, cancer and neurodegenerative diseases) as well as on industrially biotechnological issues (bioproduction of biotin). Special emphasis will be put on biogenesis of [Fe-S] clusters. Iron sulfur clusters resulting from the association between iron and sulfur atoms are cofactors in a variety of enzymes in all living organisms. They serve in a variety of biological functions including electron-transfer, gene regulation, protein structure stabilization, redox and non-redox catalysis. Researches in the last decade have shown that in vivo, [Fe-S] biogenesis requires complex biosynthetic machineries. In E. coli two types of [Fe-S] cluster biosynthesis systems have been identified so far, namely the ISC and the SUF systems. The ISC system matures [Fe-S] proteins under normal conditions whereas the SUF system operates under iron starvation or oxidative stress. By favouring a constant cross-feeding between informations derived from biophysical, biochemical, genetic, structural and bioinformatic approaches we will pursue the following objectives: (i) to provide a detailed mechanistic, structural and functional analysis of the SUF system, which should allow us to reconstitute for the first time a complete [Fe-S] biosynthesis machinery; (ii) to address the issue of specificity vs redundancy both at the biochemical, the regulatory and evolutionary levels. The occurrence of multiple [Fe-S] biogenesis systems in most organisms is calling for clarification of their role and differences; (iii) to carry out a thorough genomic and phylogenic analysis of all components involved in [Fe-S] biogenesis such as to identify putative new factors and to unfold the evolutionary history of those machineries. This history will be confronted to the arising of oxygen. A second part of this project aims at studying an, as yet poorly understood, group of enzymes involved in the conversion of C-H to C-S bonds, during biosynthesis of sulfur-containing biomolecules. These enzymes are a subgroup of a new class of [Fe-S] enzymes, called 'Radical-SAM', which are involved in a great variety of biosynthetic pathways and metabolic reactions. Such systems are particularly interesting as they are uniquely designed to finely control the manipulation of Fe and S atoms for generating, at the same time, redox active [Fe-S] clusters and sites for transient binding of sulfur atoms and from which these atoms will be made available to 'activated' substrates. We have selected as working models the tRNA thiomethyltransferase (MiaB) enzyme (which participates in the process of tRNA modification) and the biotin synthase (BioB) which catalyzes the last step of biosynthesis of biotin. Since these enzymes contain two [Fe-S], one serving for reaction with the S-adenosylmethionine (SAM), it has been suggested that the second one would function as the active sulfur atom donor (a novel function for a cluster) even though this has never been demonstrated. Furthermore, there is still a need to find the conditions which allow these two enzymes to turnover in vitro. Despite extensive investigations on these enzymes, there are important unresolved questions that we want to address in this project: (i) the connection with the ISC, SUF or other machineries involved in sulfur transfer and cluster assembly; (ii) the possible requirement for ancillary proteins which make these systems catalytic in vivo, and the reconstitution of the whole system in vitro; (iii) the molecular mechanism of sulfur transfer, a highly controversial issue and one of the most challenging question in modern enzymology. Partners 1 and 2 occupy leading positions in the field. Such a positions result from important discoveries and original contributions. Biology of [Fe-S] clusters is a most conserved process in cell biology and bringing in the evolutive facet (partner 3) is likely to open new ways and to position us in the frontline in the post-genomic era. This collaborative effort is likely to provide new concepts and actors, and to uncover the many different strategies that cells developed to benefit from sulfur chemistry.