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

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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 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.

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Acknowledgment

We acknowledge that this project is supported by the Natural Science Foundation of Shanxi province in China (No. 031042).

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Authors and Affiliations

  1. 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

    Xueyao Zhang, Jingming Yuan, Lixi Niu & Aihua Liang

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  1. Xueyao Zhang
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  2. Jingming Yuan
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  3. Lixi Niu
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  4. Aihua Liang
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Correspondence to Jingming Yuan.

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Zhang, X., Yuan, J., Niu, L. et al. Quantitative analysis and functional evaluation of zinc ion in the d-hydantoinase from Pseudomonas putida YZ-26. Biometals 23, 71–81 (2010). https://doi.org/10.1007/s10534-009-9267-7

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  • DOI: https://doi.org/10.1007/s10534-009-9267-7

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