The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.
Biochemical analysis of Escherichia coli selenophosphate synthetase mutants. Lysine 20 is essential for catalytic activity and cysteine 17/19 for 8-azido-ATP derivatization.
