PubMed · 7524578
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B S Masters. 1994. Nitric oxide synthases: why so complex?. https://doi.org/10.1146/annurev.nu.14.070194.001023
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This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 microM. The enzyme binds FAD by a simple second-order process with Kd 0.67 microM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.
L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic 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 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 Kd values of 0.24 mM and 0.22 mM, respectively. 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.
Hemorrhagic snake venom induces apoptosis of vascular endothelial cells [S. Araki, T. Ishida, T. Yamamoto, K. Kaji, and H. Hayashi (1993) Biochem. Biophys. Res. Comm. 190 , 148-153]. We have identified that a cytotoxic substance of Korean snake venom which is responsible for the apoptosis is L-amino acid oxidase (LAO). The purified enzyme is a homodimeric glycoprotein of 110,000 and is capable of generating H2O2 by catalyzing oxidation of L-amino acid. In the presence of the enzyme, cultured L1210 cell nuclei were splitted and showed the characteristic ladder-like pattern of DNA fragmentation. The enzyme binds directly to the cell surface, thereby increasing local concentration of H2O2. However, experimental evidence suggests that the LAO-induced apoptotic mechanism is distinguished from the one caused by exogenous H2O2.