Mechanistic studies of maleamate amidohydrolase (NicF) from Bordetella bronchiseptica

The genes coding the enzymes of aerobic catabolism of nicotinic acid in the genus Bordetella , the bacterial pathogen responsible for certain respiratory diseases in mammals, have recently been discerned. A step in this 6‐step mechanism of nicotinic acid degradation to fumaric acid is the hydrolytic deamination of maleamate to malate by maleamate amidohydrolase, NicF (EC 3.5.1.107). The BB1782 gene from Bordetella bronchiseptica was cloned, expressed in E. coli, and the resulting recombinant enzyme was demonstrated to catalyze the hydrolysis of maleamate to malate by proton NMR. Kinetic analysis of the reaction by isothermal titration calorimetry established the k cat and K M to be 27 s −1 and 50 μM at pH 7.5, respectively. The K M of maleamate is observed to be dependent on the ionic strength of the solution, consistent with the development of ionic interactions in the ES complex. Inhibition studies using iodoacetamide confirm a catalytic role for cysteine in the mechanism. Viscosity effects on k cat and k cat / K M and 15 N kinetic isotope effect studies will be used to establish the rate‐limiting step of the enzyme‐catalyzed reaction. This work was supported by a grant to The College of Wooster from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.