Biochemical characterization of the promiscuous activities of rhodanese family enzymes

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Université de Lorraine
The rhodanese domain (Rhd), composed of a central  sheet flanked by  helices, is found in all major evolutionary phyla and often in several proteins encoded by the same genome. Rhd-containing enzymes are classified into four families according to the number of Rhd domains and the presence, or not, of additional domains. Some of the Rhd-containing enzymes exhibit sulfurtransferase activities and are implicated in hydrogen sulfide metabolism and/or signaling processes as sulfur carriers. These enzymes catalyze a sulfur transfer reaction from a donor substrate like 3-mercaptopyruvate or thiosulfate for 3- mercaptopyruvate sulfurtransferases or thiosulfate sulfurtransferases, respectively. Human CDC25 phosphatases which catalyze the dephosphorylation of cyclin dependent kinases, belong also to this family. The fact that these enzymes exhibit a side arsenate reductase activity suggest that the Rhd-domain represents a new example of promiscuous catalyst exhibiting sulfurtransferase, phosphatase and arsenate reductase activities in the same active site. Interestingly, all these activities rely on the presence of a Cys-containing catalytic loop, but sulfurtransferases and phosphatases mainly differ by the size and sequence of the catalytic loop which is one residue longer in the later one (CX5R vs CX4R). Moreover, MOCS3, another human sulfurtransferase, was shown to display substrate specificity for protein persulfide. In this context, my project aims to 1) characterize the promiscuous activities of rhodanese family enzymes, using the human TSTD1 sulfurtransferase and the catalytic domain of the CDC25B as model enzymes, and 2) characterize the substrate specificity of MOCS3. Using a structure-function relationship approach, I have notably shown that i) contrary to TSTD1, CDC25B displays promiscuous sulfurtransferase, phosphatase and arsenate reductase activities, and ii) this promiscuity is not exclusively rely on the size and the sequence of the catalytic loop.
Rhodanese family, sulfurtransferase, phosphatase, promiscuity, structure- function, specificity