L-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD(+). The enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor.In accordance with its catalytic activities, LASPO displays strong primary and tertiary structure similarity with the Flavin containing subunit of the proteins belonging to the succinate dehydrogenase/fumarate reductase family. The similarity extends to the active site residues, with LASPO differing from the other enzymes of the family only for the presence of a conserved glutamate (E121), which is substituted by apolar amino acids in the other enzymes. Three complementary approaches have been used to define the role of E121 in LASPO: characterization of mutants (E121A, E121Q, E121D and E121 K), investigation of the catalytic activities of WT and mutants towards substrates and substrate analogues and molecular docking studies. All mutants retain fumarate reductase activity. On the contrary, all mutants lack L-aspartate oxidase activity, although retaining the ability to bind L-aspartate (except for E121 K). These results and investigations on the oxidase activity towards substrate analogues suggest that the roles of E121 in catalysis include orienting L-aspartate in a productive binding mode and favouring proton abstraction from C2 by an active site base. Molecular docking studies of the substrate (L-aspartate), inhibitor (p-aspartate) and product (imino aspartate) in the active site of LASPO confirm that (a) the substrate/product energetically favoured orientation in the active site supports the conclusions reported above, (b) E121 interacts favourably with the charged amino group of the substrate and (c) different ligands might assume different orientations in the active site of the enzyme. (C) 2010 Elsevier Masson SAS. All rights reserved.
On the catalytic role of the active site residue E121 of E. coli L-aspartate oxidase / G. Tedeschi, S. Nonnis, B. Strumbo, G. Cruciani, E. Carosati, A. Negri. - In: BIOCHIMIE. - ISSN 0300-9084. - 92:10(2010 Oct), pp. 1335-1342.
|Titolo:||On the catalytic role of the active site residue E121 of E. coli L-aspartate oxidase|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||ott-2010|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.biochi.2010.06.015|
|Appare nelle tipologie:||01 - Articolo su periodico|