Sulfur Assimilation Targets

Characterisation of S. typhimurium sulfur assimilation targets SAT and OASS (WP3)

Prof. Andrea Mozzarelli
University of Parma

The availability of cysteine is crucial for bacteria since this amino acid is the preferential source of sulfur for all sulfur-containing biological molecules, such as methionine, biotin, thiamin and iron–sulfur clusters. Moreover, in pathogens, cysteine contributes directly or as a precursor to the maintenance of the redox state of the cell. This function is particularly critical to facultative intracellular microorganisms, such as Salmonella typhimurium (and also key gram-positive species such as Mycobacterium tuberculosis) that spend part of their life-cycle in the highly oxidizing environment of macrophages, thus supporting long-term infections. Therefore, the enzymes involved in reductive sulfate assimilation are potential drug targets. This research project will investigate the enzymes involved in final two steps of cysteine biosynthesis, serine acetyl transferase (SAT) and O-acetylserine sulfhydrylase (OASS) [Chattopadhyay et al.  Biochemistry 46, 8315-8330 (2007)]. OASS is a PLP-dependent enzyme present as two isozymes, OASS-A and OASS-B (whose expression level depends on environmental conditions). Recent investigations have demonstrated that OASS mutants display a 500-fold decrease in fitness [Turnbull et al. Microbiology 154, 3410-3419 (2008)], and sulfur reductive assimilation pathway (SRAP) enzymes are a validated target for antibiotics [Bhave et al. Infect. Disord. Drug Targets, 7, 140-158 (2007)]. We aim to express and purify SAT and the OASS isozymes and develop robust HTP assays for inhibitor screening (inhibitors obtained from WP1/WP2 ). Inhibition constants will be determined (based on the dependence of catalytic activity and fluorescent intensity changes) and the ligand-enzyme interaction will be investigated via Saturation Transfer Difference (STD) NMR. If amenable, crystals of OASS and SAT will be made in the presence of strong inhibitors in order to determine their three-dimensional structures for inhibitor optimization. OASS-A, but not OASS-B, forms a tight complex with SAT, leading to OASS inhibition. In addition, SAT is inhibited by cysteine, the OASS product. Therefore, we will also characterize the interaction between SAT and OASS-A (including structural determination of the complex) and determine the expression profiles of each enzyme in different growth conditions.

Objectives: To express, purify and characterize the enzymes SAT and OASS. To screen optimized OASS inhibitors and SAT ligands by activity assays and fluorimetric methods. To crystallize SAT and OASS in the absence and presence of inhibitors, and SAT-OASS complex. To identify the regulatory mechanisms controlling SAT, OASS and SAT-OASS activities. To investigate the expression of SRAP enzymes under different growth conditions via proteomic analysis of bacterial extracts.

Expected Results: Identification of optimized inhibitors of OASS-A and –B and novel SAT inhibitors. Understanding the regulatory mechanisms controlling the synthesis of cysteine via the interaction between SAT and OASS, and via the control of the expression of SRAP enzymes.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No  642620
European Union