title: Specific amino acids responsible for the cold adaptedness of Micrococcus antarcticus β-glucosidase BglU.
abstract: Psychrophilic enzymes display efficient activity at moderate or low temperatures (4-25 °C) and are therefore of great interest in biotechnological industries. We previously examined the crystal structure of BglU, a psychrophilic β-glucosidase from the bacterium Micrococcus antarcticus, at 2.2 Å resolution. In structural comparison and sequence alignment with mesophilic (BglB) and thermophilic (GlyTn) counterpart enzymes, BglU showed much lower contents of Pro residue and of charged amino acids (particularly positively charged) on the accessible surface area. In the present study, we investigated the roles of specific amino acid residues in the cold adaptedness of BglU. Mutagenesis assays showed that the mutations G261R and Q448P increased optimal temperature (from 25 to 40-45 °C) at the expense of low-temperature activity, but had no notable effects on maximal activity or heat lability. Mutations A368P, T383P, and A389E significantly increased optimal temperature (from 25 to 35-40 °C) and maximal activity (~1.5-fold relative to BglU). Thermostability of A368P and A389E increased slightly at 30 °C. Mutations K163P, N228P, and H301A greatly reduced enzymatic activity-almost completely in the case of H301A. Low contents of Pro, Arg, and Glu are important factors contributing to BglU's psychrophilic properties. Our findings will be useful in structure-based engineering of psychrophilic enzymes and in production of mutants suitable for a variety of industrial processes (e.g., food production, sewage treatment) at cold or moderate temperatures.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, People's Republic of China.
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Miao
authors.affiliation: Tianjin Life Science Research Center and Department of Pathogen Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China.
authors.forename: Hong-Xia
authors.initials: HX
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Qu
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, People's Republic of China. liuzhp@sun.im.ac.cn.
authors.forename: Zhi-Pei
authors.initials: ZP
authors.lastname: Liu
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003080:Cold Temperature; D004795:Enzyme Stability; D008837:Micrococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D001617:beta-Glucosidase
keywords: Amino acid residues; BglU; Cold adaptation; Mutagenesis; Psychrophilic enzymes