Functional and molecular exploration of the carbohydrate sulfatases diversity – S-PLORE

Sulfated polysaccharides found in micro- and macro-algae (i.e. agars, carrageenan etc.) or secreted by marine bacteria represent the most abundant marine biomass. Even though, they are less abundant in terrestrial organism, sulfated polysaccharides are involved in many biological pathways (e.g. glycoaminoglycans). Sulfatases represent a class of enzymes which catalyze the hydrolysis of ester sulfate groups. These enzymes are important in the remolding and the degradation of sulfated polysaccharides. Despite the huge number of predicted sulfatase genes sequenced in the frame of numerous genomic and metagenomic programs, only very few sulfatases were characterized biochemically and studied at the molecular level by crystallography.
The main bottleneck when investigating this class of enzymes lies in the difficulty of producing “active” forms in sufficient amount for biochemical and structural studies. Indeed, all the known carbohydrate sulfatases are formyl-glycine dependent sulfatases which require a post-translational modification of either the catalytic cysteine or serine amino acid. This post-translational modification is very rarely obtained, and in low amount, in classical protein expression systems.
Within this context, we propose to take advantage of the novel and innovative PLOR/pSW expression system dedicated to the production of active sulfatases that we have developed to explore the functional and molecular diversity of sulfatases. We will select by bioinformatics, overexpress and screen on a unique collection of polysaccharides, about 300 putative carbohydrate sulfatases spanning the diversity of this class of enzymes. In parallel, medium throughput crystallization experiments and determination of crystal structures of a large set of sulfatases covering the selection of sub-families will be conducted.
At the end of the project, we will provide the most advanced and detailed picture of the molecular determinants of sulfated substrate recognition.