Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with L-aspartate oxidase, the other component of the so-called 'quinolinate synthetase complex,' the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken. The results are presented in this paper. Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up. The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E. coli culture. A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented. Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from L-aspartate, dihydroxyacetone phosphate, and O2 by using only nadA and nadB gene overexpressed products. (C) 2000 Academic Press.
Cloning, overexpression and purification of E. coli quinolinate synthetase / F. Ceciliani, T. Caramori, S. Ronchi, G. Tedeschi, M. Mortarino, G. Galizzi. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - 18:1(2000), pp. 64-70.
|Titolo:||Cloning, overexpression and purification of E. coli quinolinate synthetase|
CECILIANI, FABRIZIO (Primo)
MORTARINO, MICHELE (Penultimo)
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2000|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1006/prep.1999.1153|
|Appare nelle tipologie:||01 - Articolo su periodico|