Purification and characterization of quinate (shikimate) dehydrogenase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa

Abstract

The bifunctional enzyme quinate (shikimate) dehydrogenase (quinate: NAD+ oxidoreductase, EC 1.1.1.24), which catalyzes the first reaction in the inducible quinic acid catabolic pathway of Neurospora crassa, has been purified to homogeneity. The enzyme is a monomer of 41000 daltons with an s20,w = 2.94 S. However, electrophoresis under non-denaturing conditions revealed three protein species, which have both quinate and shikimate dehydrogenase activities. The enzyme, with a single binding site for both substrates, has a Km of 0.37 mM for quinate and of 1.18 mM for shikimate, although the V is about 3-fold higher with shikimate. Essential sulphydryl groups which were not localized in the active site were detected. Thermal stability of the enzyme was greatly enhanced by low concentrations of quinate, shikimate, NADH, or by high ionic strength.