title: Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics: new insights into relationship between conformational flexibility and hydrogen bonding.
abstract: Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
authors.affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Bin-Bin
authors.initials: BB
authors.lastname: Xie
authors.forename: Fei
authors.initials: F
authors.lastname: Bian
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Chen
authors.forename: Hai-Lun
authors.initials: HL
authors.lastname: He
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.forename: Yin-Xin
authors.initials: YX
authors.lastname: Zeng
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Bai-Cheng
authors.initials: BC
authors.lastname: Zhou
authors.forename: Yu-Zhong
authors.initials: YZ
authors.lastname: Zhang
mesh_terms: D000220:Adaptation, Biological; D000595:Amino Acid Sequence; D001110:Arctic Regions; D055162:Biocatalysis; D003080:Cold Temperature; D019295:Computational Biology; D003198:Computer Simulation; D017124:Conserved Sequence; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D046448:Ice Cover / Q000382:microbiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009792:Oceans and Seas; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013820:Thermolysin / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D013820:Thermolysin; D015032:Zinc