Engineering the substrate specificity of Staphylococcurs aureus sortase A -: The β6/β7 loop from SrtB confers NPQTN recognition to SrtA
Engineering the substrate specificity of Staphylococcurs aureus sortase A -: The β6/β7 loop from SrtB confers NPQTN recognition to SrtA
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DOI：
10.1074/jbc.m610519200
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发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
McCafferty, Dewey G.
中科院分区：
文献类型：
--
作者：
Bentley, Matthew L.;Gaweska, Helena;McCafferty, Dewey G.
金黄色葡萄球菌转肽酶分选酶A（SrtA）将毒力和定殖相关的表面蛋白锚定到细胞壁。SrtA选择性识别C-末端LPXTG基序，而相关的转肽酶分选酶B（Srt B）识别C-末端NPQTN基序。在这两种酶中，裂解发生在保守的苏氨酸之后，随后在苏氨酸和细胞壁肽聚糖的五甘氨酸交叉桥之间形成酰胺键。遗传和生物化学研究强烈表明，SrtA和SrtB表现出精致的识别基序的特异性。为了更好地了解这两种亚型内底物特异性的起源，我们使用序列和结构分析来预测可能参与赋予底物特异性的残基和结构域。进行突变分析和结构域交换实验以测试它们在底物识别和特异性中的功能。通过用SrtB的相应结构域替换SrtA中的β 6/β 7环，获得SrtA特异性谱的显著变化。嵌合β 6/β 7环交换酶（SrtLS）赋予了酰化含NPQTN底物的能力，k（cat）/K-mapp为0.0062 +/- 0.003 M-1 s（-1）。这种酶不能进行反应的转肽阶段，表明转肽作用需要额外的结构域。SrtLS的总体催化特异性曲线（k（cat）/K-m（app）（NPQTN）/k（cat）/K-m（app）（LPETG））与SrtA相比改变了700，000倍。这些结果表明β 6/β 7环是分选酶中底物识别的重要位点。
The Staphylococcus aureus transpepticlase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the beta 6/beta 7 loop in SrtA with the corresponding domain from SrtB. The chimeric beta 6/beta 7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a k(cat)/K-mapp of 0.0062 +/- 0.003 M-1 s(-1). This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpepticlation to occur. The overall catalytic specificity profile (k(cat)/K-m(app) (NPQTN)/k(cat)/K-m(app) (LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the beta 6/beta 7 loop is an important site for substrate recognition in sortases.