title: Improvement of the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by engineering C-terminal amino acids.
abstract: In the current study, a three-tiered mutagenesis strategy was employed to simultaneously improve the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) by engineering the last ten amino acids (Met245∼Glu254) of its C-terminal region. Initially, truncated mutagenesis results displayed that C-terminal deletions decreased the thermostability and/or activity of HheC. Then ten residues were subjected to single-site saturation mutagenesis, resulting in 20 beneficial single-point variants related to the thermostability or activity of HheC. The results clearly indicated that residues Met252∼Glu254 and Trp249 are crucial for regulating enzyme thermostability and activity, respectively. Finally, the beneficial substitutions were combined using efficient multi-site combinatorial mutagenesis approaches, leading to an outstanding variant PX14 (Trp249Pro/Met252Leu/Pro253Asp), which had a 17.8-fold higher half-life and a 4.0-fold higher kcat value than that of wild-type HheC. These results indicated that the C-terminal residues play an important role in modulating both the thermostability and activity of HheC.
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. Electronic address: wangxiong365436@163.com.
authors: Xiong
authors: X
authors: Wang
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. Electronic address: shaoqianghan@163.com.
authors: Shaoqiang
authors: S
authors: Han
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. Electronic address: yangzujun@uestc.edu.cn.
authors: Zujun
authors: Z
authors: Yang
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. Electronic address: lixiatang@uestc.edu.cn.
authors: Lixia
authors: L
authors: Tang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016415:Sequence Alignment
chemical_list: D000596:Amino Acids; D006867:Hydrolases; C430848:halohydrin dehalogenase
keywords: C-terminal; Halohydrin dehalogenase; Multi-site combinatorial mutagenesis; Single-site saturation mutagenesis; Truncated mutagenesis