Probing the active site of L-aspartate oxidase by site-directed mutagenesis: Role of basic residues in fumarate reduction

L-Aspartate oxidase is a very particular oxidase which behaves as a fumarat e reductase in anaerobic conditions. Its primary and tertiary structures pr esent remarkable similarity with the soluble fumarate reductase (FRD) from Shewanella frigidimarina and the flavin subunit of the membrane-bound fumar ate reductase from Escherichia coli and Wolinella succinogenes. This and ot her extensive similarities are consistent with the idea that a common catal ytic mechanism for the reduction of fumarate operates for all members of th is enzyme group and that the key residues involved in the substrate binding and catalysis are conserved. This manuscript reports information about the role of these basic residues in L-aspartate oxidase: R290, R386, H244, and H351. By means of site-directed mutagenesis, R290 and R386 are mutated to Leu and H351 and H244 are mutated both to Ala and Ser. H351, H244, and R386 mutants bind substrate analogues with higher dissociation constants and pr esent lower k(cat)/K-m values in the reduction of fumarate. Therefore, the results indicate that R386, H244, and H351 are important for the binding of the substrate fumarate and may play an important but not essential role in catalysis. R290, on the contrary, is mainly involved in catalysis and not in substrate binding since its mutation abolishes the catalytic activity wi thout lowering the affinity of the enzyme for the substrate. The redox prop erties of all the mutants are identical to the wild-type. The findings are consistent with a model of L-aspartate oxidase active site based on the hyp othesis proposed for the soluble FRD from S. fridimarina.