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1: Protein Expr Purif. 2000 Feb;18(1):64-70.

Cloning, overexpression, and purification of Escherichia coli quinolinate
synthetase.

Ceciliani F, Caramori T, Ronchi S, Tedeschi G, Mortarino M, Galizzi A.

Istituto di Fisiologia Veterinaria e Biochimica, UniversitУљ di Milano, Milan,
Italy.

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 O(2) by using only nadA and nadB
gene overexpressed products. Copyright 2000 Academic Press.