Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26

The imidase from Pseudomonas putida YZ-26 consisting of 293-amino acid residues is a novel imidase with four subunits as the holo-enzyme and low molecular weight which is significantly different from known mammalian imidase. This study measured the zinc-binding properties of the imidase using inductively coupled plasma-atomic emission spectrometry and competition assay combined with activity determinations. Results show that each subunit of the imidase binds the zinc ion by 1:1 stoichiometry with apparent binding constant of 9.5 × 108 M−1. The activity of the apo-imidase (20 μM) was recovered with the addition of zinc in the lower concentration (0–20 μM), whereas the enzymatic activity is decreased in the presence of high concentration of zinc (above 100 μM). The site-directed mutagenesis of His247, His86 or Cys7, Cys108 in imidase resulted in loss of activity and zinc-binding abilities at different degrees, showing that these residues may critically affect both enzymatic activity and conformation.