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.
Highlights
Cold-adapted enzymes, produced by organisms thriving in permanently cold habitats, are characterized by high catalytic efficiencies at low temperatures and low stabilities at high temperatures [1,2,3,4]
Cold Adaptation of Zinc Metalloproteases pseudolysin is from the mesophilic bacterium Pseudomonas aeruginosa PAO1, and a putative vibriolysin in this family is from the Antarctic bacterium strain 643
As shown in the results, these three enzymes made up a good system to study the structural basis underlying cold adaptation, because (i) the three enzymes are from typical habitats and the comparison of two enzymes at different cold-adaptation levels may provide more information than just comparing a cold-adapted enzyme with a mesophilic enzyme, and (ii) these three enzymes are highly homologous, allowing an easy interpretation of the contributions of different structural features to cold adaptation
Summary
SM495 was isolated from a sea ice core sample 122–124 cm deep from the ice surface, which was taken from the Canadian Basin, Arctic Ocean (sampling site: 75°28Ј52ЉN 152°51Ј18ЉW) during the 2nd Chinese National Arctic Research Expedition, summer of 2003. The P. aeruginosa PAO1 strain was a generous gift from Yu Li-Yan (National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical Col-
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