Bentley Matthew L, Gaweska Helena, Kielec Joseph M, McCafferty Dewey G
Department of Biochemistry and Biophysics and the Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2007 Mar 2;282(9):6571-81. doi: 10.1074/jbc.M610519200. Epub 2007 Jan 2.
The Staphylococcus aureus transpeptidase 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 beta6/beta7 loop in SrtA with the corresponding domain from SrtB. The chimeric beta6/beta7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a k(cat)/K(m)(app) 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 transpeptidation 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 beta6/beta7 loop is an important site for substrate recognition in sortases.
金黄色葡萄球菌转肽酶分选酶A（SrtA）将与毒力和定植相关的表面蛋白锚定到细胞壁上。SrtA选择性识别C末端LPXTG基序，而相关的转肽酶分选酶B（SrtB）识别C末端NPQTN基序。在这两种酶中，切割发生在保守的苏氨酸之后，随后苏氨酸与细胞壁肽聚糖的五甘氨酸交联桥之间形成酰胺键。遗传和生化研究有力地表明，SrtA和SrtB对其识别基序表现出极高的特异性。为了更好地理解这两种同工型底物特异性的起源，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。进行了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β6/β7环，获得了SrtA特异性谱的显著变化。嵌合的β6/β7环交换酶（SrtLS）赋予了酰化含NPQTN底物的能力，其k(cat)/K(m)(app)为0.0062±0.003 m(-1) s(-1)。该酶无法进行反应的转肽阶段，这表明转肽发生还需要其他结构域。SrtLS的总体催化特异性谱（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，β6/β7环是分选酶中底物识别的重要位点。
