Probing the role Cysteine-150 in Maleamate Amidohydrolase (NicF) catalysis from Bordetella bronchiseptica RB50 by site-directed mutagenesis

Nicholas Spittle, The College of Wooster

Abstract

Growing resistance of bacteria to current antibiotics makes research into new antibiotic compounds and methods more important. Also, the need for inexpensive and effective methods to degrade harmful, potential cancer-causing chemicals in the environment is on the rise. The genes coding for the enzymes involved in the aerobic degradation of nicotinic acid in the bacterial genus Bordetella have been recently characterized. The fifth step of this catabolic pathway involves hydrolytic deamidation of maleamate into maleate by maleamate amidohydrolase, NicF. The goal of this study was to probe the role of a particular amino acid residue by site-directed mutagenesis. Cys150, identified by sequence homology of enzymes with known catalytic mechanisms and spatial orientation of a NicF crystal structure, is hypothesized to be a vital residue in the mechanism of NicF, likely acting as a nucleophile. Upon mutation of Cys→Ser, a significant reduction in catalytic activity was observed. Catalysis was observed when analyzing production formation using 1H NMR and a UV GDH linked assay. MMTS, a molecule shown to inhibit NicF activity by binding to the thiol side chain of Cys150, was then used to inhibit possible wild-type contamination. Inhibition of wild-type contaminants with MMTS resulted in no observable rate difference between a C150S NicF-catalyzed reaction and an uncatalyzed reaction, suggesting that most, if not all, catalytic activity observed from the mutant enzyme was due to wild-type contamination. Thus, these findings suggest the role of Cys150 in the catalytic mechanism of NicF appears to be critical, with Cys150 likely acting as the first nucleophile in the mechanism.