Eur. J. Biochem. zyxwvutsrqpo 239, 427-433 (1996) zyxwvuts 0 FEBS 1996 zyxwvutsrqpo L-Aspartate oxidase from zyxwv Escherichia coli 11. Interaction with C, dicarboxylic acids and identification of a novel L-aspartate zyxwvu : fumarate oxidoreductase activity Gabriella TEDESCHI, Armando NEGRI, Michele MORTARINO, Fabrizio CECILIANI, Tatjana SIMONIC, Ludovica FAOTTO and Severino RONCHI Istituto di Fisiologia Veterinaria e Biochimica, UniversitB di Milano and Centro Interuniversitario per lo Studio delle Macromolecole Informazionali, Italy (Received 6 MarcN17 May 1996) - EJB 96 0313/4 L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosyn- thetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia zyxwvuts coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with K,, values of 0.24 mM and 0.22 mM, respec- tively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate :fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase. Keywords: flavoprotein; L-aspartate oxidase ; FAD ; fumarate reductase ; photoreduction. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol non-covalently bound FAD/mol protein. The molecule catalyzes the oxidation of L-aspartate to the corresponding i m - noacid and the reduction of oxygen to H,O, zyxwvut [I]. In E. coli, the enzyme is specified by the nadB gene and is one of the two components of the quinolinate synthase complex. The complex catalyzes the biosynthesis of quinolinate from L-aspartate and dihydroxyacetone phosphate in the de zyxwvutsr now biosynthetic path- way for pyridine nucleotide formation [2]. Quinolinate is subse- quently converted to NAD via a metabolic sequence common to all organisms. The de now NAD biosynthesis has been termed an anaerobic pathway based upon the observation that a muta- tion in the gene coding for L-aspartate oxidase is expressed un- der both aerobic and anaerobic growth conditions [3]. In particu- lar, although in vitro studies demonstrate that oxygen is the obli- gate electron acceptor for L-aspartate oxidase [I], the possibility was not excluded that in vivo, under anaerobic conditions, L- aspartate oxidase utilizes an electron acceptor other than oxygen. However, as far as we know, this compound is still undefined. In facultative aerobic organisms like E. coli, fumaric acid can be utilized as the terminal electron acceptor for biological oxidation of various organic compounds. The succinate-fuma- rate couple plays a role as either oxidant or reductant for the respiratory chain. The two reactions are catalyzed by succinate dehydrogenase and fumarate reductase, whose flavin-containing subunits have striking structural similarities with L-aspartate oxi- dase [4]. In particular, as already indicated in the accompanying manuscript, stretches of amino acid sequence involved in coen- zyme binding and residues thought to contribute to the active site of fumarate reductase and succinate dehydrogenase are also well conserved in L-aspartate oxidase from E. coli and from B. subtilis. Therefore, we were interested in finding if this homol- ogy extends to the properties of the active site of these enzymes. In this paper, we suggest that fumarate and succinate can bind to L-aspartate oxidase at or close to the active site of the enzyme. Two so far unknown catalytic activities of L-aspartate oxidase are reported: a fumarate reductase activity, which uses reduced benzylviologen as electron donor, and an L-aspartate :fumarate oxidoreductase activity. Steady-state kinetic parameters of the latter activity are compared with those obtained using oxygen as electron acceptor. Moreover, the stoichiometry and identification of the product of the L-aspartate:fumarate oxidoreductase activ- ity are reported in detail. Based upon these observations, a pos- sible role of fumarate as an alternative substrate to oxygen for reduced L-aspartate oxidase is proposed and its physiological relevance is discussed. Correspondence to S. Ronchi, Istituto di Fisiologia Veterinaria e Bio- Fax: +39 2 2666301. Enzyme. L-Aspartate oxidase (EC 1.4.3.16); malate dehydrogenase chimica, UniversitB di Milano, via Celoria 10, 1-20133 Milano, Italy (EC 1.1.1.37); xanthine oxidase (EC 1.1.3.22); catalase (EC 1.11.1.6). MATERIALS AND METHODS L-Aspartate oxidase overexpressed in E. coli was purified according to the procedure described in the accompanying