Quantitative analysis and functional evaluation of zinc ion in the d-hydantoinase from Pseudomonas putida YZ-26

d-Hydantoinase (HDT) is a metal-dependent enzyme that is widely used in industrial bioconversion to d-amino acids as valuable intermediates in the fields of food, pharmaceutical industry and agriculture. In this report, we prepared apo-HDT (metal-removed HDT) and Zn2+-HDT (Zn2+-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn2+-HDT and re-HDT contain 2.17 and 0.95 mol Zn2+ per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn2+ per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl2, the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn2+-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn2+-binding sites, one is an intrinsic or tight-binding site (zinc-α) essential for its activity and the other is a vacant or loose-binding site (zinc-β) possibly non-essential for the activity.