Investigation into the catalytic mechanism of Maleamate Amidohydrolase (NicF) from Bordetella bronchiseptica RB50
The oxidative degradation of nicotinate (vitamin B3) to fumarate is considered a model system for the bacterial degradation of N-heterocyclic compounds, some of which are major pollutants; yet, only recently has the complete set of genes corresponding to the enzymes involved in this pathway been identified. Of the six enzymes involved in the degradation of nicotinate, maleamate amidohydrolase (NicF), is a hydrolytic deamidase involved in the penultimate reaction of the pathway: the hydrolytic deamination of maleamate to maleate. The goal of this study was to kinetically and mechanistically characterize NicF. A kinetic inhibition assays using maleate established that the binding affinity of maleate by NicF is ~ 100-fold weaker than that of maleamate. Additionally, the highly conserved cysteine residue, Cys150, from the catalytic triad was shown to be crucial for catalysis through the use of the thiol-blocking reagent, MMTS. Additionally, an analysis of the effect of the salts, NaF and NaCl, on NicF activity led to the conclusions that cationic amino acids, likely Arg175 and Lys190, are important for the binding of maleamate. Furthermore, an examination of the use of the substrate, succinamate, suggested that the hypothesized catalytically important residues, Arg175 and Lys190, may induce NicF*maleamate binding. A more detailed understanding of the mechanism of NicF has the potential to reveal information helpful in managing environmental waste concerns as well as infectious diseases.
© Copyright 2012 Jeffrey Noel