Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation
Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation
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DOI：
10.1093/protein/gzm006
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发表时间：
2007-03-01
影响因子：
2.4
通讯作者：
Kanaya, S.
中科院分区：
文献类型：
--
作者：
Pulido, M. A.;Koga, Y.;Kanaya, S.
来自超嗜热古菌Thermococcus kodakaraensis的Tk-枯草杆菌蛋白酶以前体形式（prepro-Tk-枯草杆菌蛋白酶）合成，以前体形式（pro-Tk-枯草杆菌蛋白酶）分泌，并在前肽[Tk-前肽; Tk-枯草杆菌蛋白酶的前肽（Gly 1-Leu 69）]的自动加工和降解后成熟为活性形式（mat-Tk-枯草杆菌蛋白酶 *; Tk-枯草杆菌蛋白酶成熟结构域的Ca 2+结合活性形式）。Pro-Tk-枯草杆菌蛋白酶仅在80 ° C下显示出形成晕圈的活性，而在70和60 ° C下不显示出形成晕圈的活性，因为Tk-前肽不能被mat-Tk-枯草杆菌蛋白酶 * 有效地降解，并且在< 80 ° C下与mat-Tk-枯草杆菌蛋白酶 * 形成无活性的复合物。在整个prepro-Tk-枯草杆菌蛋白酶基因中进行随机诱变，然后筛选在70和60摄氏度下具有晕形成活性的突变蛋白质，使我们能够鉴定负责pro-Tk-枯草杆菌蛋白酶低温适应的前肽区域中的单个Gly 56-> Ser突变。SDS-PAGE分析和mat-Tk-subtilisin* 活性测定表明pro-G56 S-subtilisin比pro-Tk-subtilisin成熟更快。所得的活性形式在活性和稳定性上与mat-Tk-枯草杆菌蛋白酶 * 没有区别，表明Gly 56-> Ser突变不会严重影响成熟结构域的折叠。然而，这种突变极大地破坏了前肽的稳定性，使其以分离的形式非结构化。因此，具有Gly 56-> Ser突变的Tk-前肽（G56 S-前肽）比Tk-前肽更容易被蛋白水解降解，并且更不有效地抑制mat-Tk-枯草杆菌蛋白酶 * 活性。这些结果表明，pro-G56 S-枯草杆菌蛋白酶在较低温度下比pro-Tk-枯草杆菌蛋白酶更有效地成熟，因为自加工的G56 S-前肽在从mat-Tk-枯草杆菌蛋白酶 * 解离时是非结构化的，因此被mat-Tk-枯草杆菌蛋白酶 * 有效地降解。
Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca2+-bound active form of matured domain of Tk- subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80 degrees C, but not at 70 and 60 degrees C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at < 80 degrees C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60 degrees C, allowed us to identify single Gly56 -> Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS-PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 -> Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 -> Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.