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The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited

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Abstract

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of d-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

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Acknowledgments

The authors wish to thank the National Science Foundation of Shanxi Province (NSFSX, 031042), China for this research support. We gratefully acknowledge the valuable discussion with Dr. Tao Yuan from Sanofi Pasteur, Canada.

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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, Taiyuan, 030006, People’s Republic of China

    Xue-Yao Zhang, Li-Xi Niu, Ya-Wei Shi & Jing-Ming Yuan

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  1. Xue-Yao Zhang
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  2. Li-Xi Niu
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  4. Jing-Ming Yuan
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Correspondence to Jing-Ming Yuan.

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Zhang, XY., Niu, LX., Shi, YW. et al. The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited. Appl Biochem Biotechnol 144, 237–247 (2008). https://doi.org/10.1007/s12010-007-8004-3

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