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
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
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DOI：
10.1074/jbc.m808421200
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发表时间：
2009-04-03
影响因子：
4.8
通讯作者：
Zhang, Yu-Zhong
中科院分区：
文献类型：
--
作者：
Xie, Bin-Bin;Bian, Fei;Zhang, Yu-Zhong
构象灵活性的增加是低温下低温酶高催化效率的主要原因。然而，人们对灵活性的结构性决定因素知之甚少。我们报道了深海细菌的溶弧素MCP-02和北冰洋细菌的溶弧素E495这两个新的耐寒锌金属蛋白水解酶，并与它们的中温同源物--陆生细菌的假溶血素进行了比较。它们的催化效率k(CAT)/K-m(10-40℃)顺序为：伪溶素<；MCP-02<；E495，比例接近1：2：4。MCP-02和E495具有相同的最适温度(T-opt，比拟溶血素低57℃，比拟溶血素低5℃)和表观熔融温度(T-m=℃，相当于比拟溶血素低10℃)。结构分析表明，由于盐桥数量的减少，稳定性略有下降。荧光猝灭实验和分子动力学模拟表明，该蛋白质的柔韧性为假溶血素<；MCP-02<；E495，表明柔韧性的优化是一种冷适应策略。分子动力学结果表明，从伪溶素到MCP-02和E495的柔韧性依次增加，特别是从MCP-02到E495的增加，主要是由于天冬氨酸、丝氨酸和苏氨酸残基的增加导致了动态结构中氢键稳定性的降低。最后，提出了MCP-02和E495的冷适应模型。这是将氢键动力学优化作为冷适应策略的第一份报告，并为构象灵活性的结构基础提供了新的见解。
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 similar to 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, similar to 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.