Structural Evolution of Enzymes from Antarctic Psychrotrophs and Molecular Mechanism for Their Higher Activities at Low Temperatures

南极耐冷生物酶的结构演化及其低温高活性的分子机制

• 批准号: 13660096
• 负责人: WATANABE Kenichi
• 金额: $ 2.3万
• 依托单位: Saga University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660096/
• 关键词: subtilisin; cold-activity; cold-enzyme; psychrotroph; protease; stability of protein; structural evolution; cold-adaptation; βターン; タンパク質変性

Enzymes produced by cold-adapted microorganisms generally exhibit higher catalytic efficiency at low temperatures and lower thermostability than their mesophilic counterparts. We here report on the subtilisin-like alkaline serine protease Apa2 secreted by Antarctic psychrotroph Alteromonas sp. AS-11, which is characterized by not only a higher proteolytic activity at low temperatures but also a greater thermostability than the mesophilic subtilisin BPN'. The specific proteolytic activity of Apa2 relative to subtilisin BPN' increased with decreasing temperature, although the optimal temperatures of both enzymes were the same. At 0℃, Apa2 was 9.9-fold more active than subtilisin BPN'. It had 10 times longer half-life time at 57℃ than subtilisin BPN' with higher activation enthalpy and entropy for thermal denaturation. These features of Apa2 support the idea that structural determinants , of conformational flexibility required for high catalytic efficiency can be separated from those of thermal instability. The gene apa2 encodes a 629-amino-acid pre-pro-peptide containing a mature enzyme of 283 residues that has significant homologies with subtilisin-like serine proteases. The structural model of Apa2 suggests that the disulfide bonds, a higher number of salt bridges, and Ca^<2+>binding contribute to its thermostability, while its cold-adaptation may be attributed to the increase in structural flexibility at loop and β-turn or bend regions:

由冷适应微生物产生的酶通常在低温下表现出更高的催化效率和比它们的嗜中温对应物更低的热稳定性。本文报道了由南极嗜冷菌Alteromonas sp. AS-11分泌的类枯草杆菌蛋白酶碱性丝氨酸蛋白酶Apa 2，它不仅在低温下具有较高的蛋白水解活性，而且比嗜中温枯草杆菌蛋白酶BPN '具有更高的热稳定性。Apa 2相对于枯草杆菌蛋白酶BPN'的比蛋白水解活性随着温度的降低而增加，尽管两种酶的最适温度是相同的。在0℃下，Apa 2的活性是枯草杆菌蛋白酶BPN '的9.9倍。它在57℃的半衰期比枯草杆菌蛋白酶BPN'长10倍，热变性的活化焓和熵也更高。Apa 2的这些特征支持这样的想法，即高催化效率所需的构象灵活性的结构决定因素可以与热不稳定性的结构决定因素分离。基因apa 2编码一个629个氨基酸的前原肽，含有一个283个残基的成熟酶，与枯草杆菌蛋白酶样丝氨酸蛋白酶具有显着的同源性。Apa 2的结构模型表明，二硫键、较高数量的盐桥和Ca^<2+>结合有助于其热稳定性，而其冷适应可能归因于环和β-转角或弯曲区域的结构柔性的增加：

项目成果

Eijiro Honjo: "Genomic clones encoding two isoforms of pokeweed antiviral protein in seeds (PAP-S1 and S2) and N-glycosidase activities"Journal of Biochemistry. 131(2). 225-231 (2002)

Eijiro Honjo：“编码种子中美洲商陆抗病毒蛋白两种亚型（PAP-S1 和 S2）的基因组克隆和 N-糖苷酶活性”《生物化学杂志》。

Keiichi Watanabe: "A new crystal form of the NKl splice variant of HGF/SF demonstrates extensive hinge movement and suggests dimer formation by domain swapping"Journal of Molecular Biology. (in press). (2002)

Keiichi Watanabe：“HGF/SF NK1 剪接变体的新晶体形式表现出广泛的铰链运动，并表明通过结构域交换形成二聚体”《分子生物学杂志》。

Keiichi Watanabe: "A-new crystal form of the NK1 splice Variant of HGF/SF demonstrates extensive hinge movement and suggests that the NK1 dimer originates by domain swapping"Journal of Molecular Biology. 319. 283-288 (2002)

Keiichi Watanabe：“HGF/SF NK1 剪接变体的一种新晶体形式表现出广泛的铰链运动，并表明 NK1 二聚体源自结构域交换”《分子生物学杂志》。

Eijiro Honjo: "Real-time kinetic analyses of the interaction of ricin toxin A-chain with ribosome prove a conformational change involved in complex formation"Journal of Biochemistry. 131(2). 267-275 (2002)

Eijiro Honjo：“蓖麻毒素 A 链与核糖体相互作用的实时动力学分析证明了复合物形成中涉及的构象变化”《生物化学杂志》。

Eijiro Honjo: "Genomic clones encoding two isoforms of pokeweed antiviral protein in seeds (PAP-S1 and S2) and N-glycosidase activities"Journal of Biochemistry. 131. 225-231 (2002)

Eijiro Honjo：“编码种子中美洲商陆抗病毒蛋白两种亚型（PAP-S1 和 S2）的基因组克隆和 N-糖苷酶活性”《生物化学杂志》。