Oxalate decarboxylase (OxDc) catalyzes the conversion of oxalate to fumarate in the presence of Mn2 ion. The functional unit of OxDc from Bacillus subtilis is a hexamer with each monomer harboring two Mn2 ions coordinated by spatially close histidine residues within a large cavity. Apart from these histidines, there are 8 other histidine residues and several Asp/Glu residues distributed throughout the structure that potentially determine the pH sensitivity of OxDc in the pH range 5-8.

pH dependent stability in proteins is determined by the overall electrostatic interaction energy changes between charged residues determined by their respective pKa values. Accordingly, we expect to address the pH dependent stability (could be local stability) that is linked to activity by combinatorially generating hundreds of thousands of mutations of charged residues employing the charge-shuffling procedure that eliminates electrostatic frustration. We have developed an algorithm that does this in silico using a single structure as input. An experimentally consistent web-server that does this is in scalable manner is already available for smaller proteins (http://pbl.biotech.iitm.ac.in/pStab).

Since OxDc is a larger protein (at least 372 residues as per the PDB structure 1UW8), the number of pairwise interactions increases exponentially requiring us to tune the algorithm. We expect the modified algorithm to more robustly sample the mutational space (up till pentuple mutants). The charge-shuffling procedure will not mutate residues at the interface, active-site residues or residues that already exhibit significantly favorable charge-charge interaction energy. The genes corresponding to the mutants we provide as deliverables can then be synthesized, protein purified and characterized.

To identify single- and/or multiple-point mutations in silico that can shift the catalytically optimum pH of Oxalate decarboxylase (Bacillus subtilis) from pH 5 to pH 7.