Structure of L-aspartate oxidase: implications for the succinate dehydrogenase/fumarate reductase oxidoreductase family

Background: Given the vital role of NAD(+) in cell metabolism, the enzymes involved in bacterial de novo NAD(+) biosynthesis are possible targets for drug design against pathogenic bacteria, The first reaction in the pathway is catalysed by L-aspartate oxidase (LASPO), a flavoenzyme that converts as partate to iminoaspartate using either molecular oxygen or fumarate as elec tron accepters, LASPO has considerable sequence homology with the flavoprot ein subunits of succinate dehydrogenase (SDH) and fumarate reductase (FRD). Results: The crystal structure of the apoform of LASPO from Escherichia col i has been determined to 2.2 Angstrom resolution, The enzyme shows a novel fold for an FAD-dependent protein, comprising a three-domain structure: an FAD-binding domain with the dinucleotide-binding fold, a C-terminal three-h elical bundle domain, and an alpha+beta capping domain, which is topologica lly similar to the small subunit of spinach ribulose-1,5-bisphosphate carbo xylase/oxygenase. The interface between the PAD-binding and capping domains defines a cleft in which the active site is located. Conclusions: A number of strictly conserved residues present in all three d omains indicate that LASPO, SDH and FRD share the same overall folding topo logy. Many of these conserved residues are in the PAD-binding site and acti ve centre, suggesting a similar catalytic mechanism. Thus, LASPO, SDH and F RD form a class of functionally and structurally related oxidoreductases th at are all able to reduce fumarate and to oxidise a dicarboxylate substrate .