Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase

Quinolinate synthetase catalyzes the second step of the de novo biosyntheti c pathway of pyridine nucleotide formation. In particular, quinolinate synt hetase is involved in the condensation of dihydroxyacetone phosphate and im inoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with L-aspartate oxidase, th e other component of the so-called "quinolinate synthetase complex," the cl oning, the overexpression, and the purification to homogeneity of Escherich ia coli quinolinate synthetase were undertaken. The results are presented i n this paper. Since the overexpression of the enzyme resulted in the format ion of inclusion bodies, a procedure of renaturation and refolding had to b e set up. The overexpression and purification procedure reported in this pa per allowed the isolation of 12 mg of electrophoretically homogeneous quino linate synthetase from 1 liter off. 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 ot her enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from L-aspartate, dihyd roxyacetone phosphate, and O-2 by using only nadA and nadB gene overexpress ed products. (C) 2000 Academic Press.