The Multiple Roles of the Active Site Loop of Watermelon Glyoxysomal Malate Dehydrogenase

Abstract

All malate dehydrogenases have a flexible loop that starts to close over the active site region of malate dehydrogenase when cofactor binds and locks down completing the active site when the dicarboxylic acid substrate binds. As such the active site loop appears to play a role in both substrate specificity and correct alignment of the substrate to allow hydride transfer and proton abstraction/donation depending on the direction of the reaction. Combined with available crystal structures, detailed analysis of the 1smk.pdb structure shows the loop in a series of microstates from fully open to a closed state induced by the inhibitor citrate binding (figure 1), sequence conservation suggests that the loop region contains a 19 amino acid stretch from P119 to N137 using the watermelon glyoxysomal enzyme as reference. Clustal Omega Analysis of malate dehydrogenases from prokaryotes to mammals shows 5 families of conservation of the loop region (figure 2) allowing a series of hypotheses about structure function relationships of specific residues within the sequence to be made which have been tested by a combination of computational and site directed mutagenesis wetlab approaches. In addition to the two canonical arginines involved in substrate binding, we have explored every other residue in the loop region. Overall, mutations in the first half of the loop had small impact on Specific Activity while mutations in the second half of the loop had larger impacts. R130 mutants to E or S had the most impact while mutation to A or Q had lesser effects. Three other residues in particular had striking effects on Oxaloacetate saturation, G121A, L133S and N137. L133 and N137, at the C terminal base of the loop, were hypothesized to play a role in the repulsive interactions between substrate and cofactor in the active site. Consistent with this hypothesis, creation of an L133S or D137A mutant showed that concomitant with a reduction in specific activity to 2% and 5% wildtype (respectively), Km for Oxaloacetate was dramatically increased while Km for NADH was decreased somewhat. Finally, a series of mutations of M128 (Q,I and A) were explored with varying effects on specific activity (Q>I>A) and significant effects on Km for Oxaloacetate but lesser or no effects on Km for NADH. The impact of M128 mutations in particular were further explored computationally using DeepDDG analysis and effects on stability correlated with impact on kinetic properties. Overall these studies provide a detailed picture of the complex interactions the flexible loop makes that contribute to both substrate binding and catalysis.