Quinolinate Synthase "complex" in Bacillus subtilis

The need of NAD(P) in many microrganisms, including several pathogens, depends on two processes: de novo biosynthesis and recycle of NAD metabolites. The first two steps in NAD de novo biosynthesis lead to the production of quinolinate starting from L-aspartate, dihydroxyacetonephosphate and an oxidant (oxygen, fumarate or quinones in vitro) catalyzed by NadB (L-aspartate oxidase) and NadA (quinolinate synthase).These two enzymes - which are sometimes proposed as being involved in a multienzyme complex - are absent in mammalian; therefore they are considered ideal targets for the development of novel prophylactic and therapeutic agents. For such purpose it is of the utmost importance to gain a thorough knowledge of both their biochemical and structural properties and of the factors controlling their expression and functionality in vivo. This communication reports studies aimed at the biochemical characterization of recombinant NadA and NadB from B. subtilis overexpressed in E.coli and the investigation of the possible existence of the putative complex between the two enzymes. Recombinant catalytically active NadA was obtained following purification in strictly anaerobic conditions and used to determine relevant biochemical properties, including the stability of the enzyme towards oxygen either in the absence or in the presence of NadB, the kinetic parameters for the reaction catalysed by NadA both under aerobic and anaerobic conditions and the characterization of its of Fe/S center. Studies on NadB, including coenzyme and selected ligands affinity and kinetics parameters for the various catalytic activities, allowed to conclude that the properties of the enzyme from B. subtilis are similar to those of the E. coli enzyme previously characterised by our group. The existence and properties of the putative NadA/NadB multienzymatic complex was investigated using both biochemical and proteomic approaches. The former included evaluation of the effect of NadA on the properties of NadB (coenzyme affinity, UV-visible absorption and fluorescence due to the flavin coenzyme, polymerization state) and vice-versa (stability of NadA towards oxygen, polymerization state); the proteomic approach was accomplished by performing affinity chromatography using each protein immobilised on suitable chromatographic media and evaluating binding of the partner protein under various experimental conditions.