title: Quantitative analysis and functional evaluation of zinc ion in the D-hydantoinase from Pseudomonas putida YZ-26.
abstract: 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 Zn(2+)-HDT (Zn(2+)-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn(2+)-HDT and re-HDT contain 2.17 and 0.95 mol Zn(2+) per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn(2+) per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl(2), the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn(2+)-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn(2+)-binding sites, one is an intrinsic or tight-binding site (zinc-alpha) essential for its activity and the other is a vacant or loose-binding site (zinc-beta) possibly non-essential for the activity.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Institute of Biotechnology, Shanxi University, 92 Wu-cheng Road, 030006 Taiyuan, People's Republic of China.
authors.forename: Xueyao
authors.initials: X
authors.lastname: Zhang
authors.forename: Jingming
authors.initials: J
authors.lastname: Yuan
authors.forename: Lixi
authors.initials: L
authors.lastname: Niu
authors.forename: Aihua
authors.initials: A
authors.lastname: Liang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002942:Circular Dichroism; D007477:Ions / Q000032:analysis; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000032:analysis*
chemical_list: D007477:Ions; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D015032:Zinc