CHARACTERIZATION OF THE TYPE-I DEHYDROQUINASE FROM SALMONELLA-TYPHI

The type I dehydroquinase from the human pathogen Salmonella typhi was overexpressed in an Escherichia coli host and purified to homogeneity . The S. typhi enzyme was characterized in terms of its kinetic parame ters, important active-site residues, thermal stability and c.d. and f luorescence properties. In all important respects, the enzyme from S. typhi behaves in a very similar fashion to the well-characterized enzy me from E. coli, including the remarkable conformational stabilization observed on reduction of the substrate/product mixture by NaBH4. This gives confidence that the information from X-ray studies on the S. ty phienzyme [Boys, Fawcett. Sawver, Moore, Charles, Hawkins, Deka, Klean thous and Coggins (1992) J. Mol. Biol. 227, 352-355] can be applied to other type I dehydroquinases. Studies of the quenching of fluorescenc e of the S. typhi enzyme by succinimide show that NaBH4 reduction of t he substrate/product imine complex involves a dramatic decrease in the flexibility of the enzyme, with only very minor changes in the overal l secondary and tertiary structure.