Interface Mutants of Glyoxasomal Malate Dehydrogenase

Subunit communications play an important role in many proteins whether homopolymers or heteropolymers. In the homopolymer glyoxasomal malate dehydrogenase we have analyzed the nature of the subunit interactions using bioinformatics approaches, and the computational algorithm HINT. Various mutants were designed to strengthen the interactions at the interface, leading to a more stable dimer of the protein. These mutations have been created using Quikchange mutagenesis and expressed. The resultant proteins have been purified using the introduced histidine tag and characterized using both structural and functional measurements. The mutants show overall structural properties consistent with similar structures to the native protein but several have significantly different kinetics properties from the native protein. The L233A mutant shows altered properties with respect to NADH saturation while the V152A mutant shows distinct differences with respect to Km for the substrate oxaloacetate. A second series of mutants involved H90 and E256. H90 extends across the subunit interface and potentially forms an ion pair with E256 on the opposite subunit. Mutation of H90 to Leucine results in an enzyme dimer which is no longer subject to either citrate activation or to substrate inhibition at excess oxaloacetate concentrations. E256Q shows similar properties while mutation of a nearby arginine to glutamine has little effect. Overall, these studies suggest both that subunit interactions play a significant role in the overall catalytic cycle of Malate Dehydrogenase and also that distinct regions of the subunit interface play specific roles in subunit communications. Structural studies of the H90L and E256Q mutants by x ray crystallography are underway. The work is supported by National Science Foundation Grant MCB 0448905 to Ellis Bell