Design of Ca2+-independent Staphylococcus aureus sortase A mutants
Design of Ca2+-independent Staphylococcus aureus sortase A mutants
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DOI：
10.1002/bit.24585
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发表时间：
2012-12-01
影响因子：
3.8
通讯作者：
Nagamune, Teruyuki
中科院分区：
文献类型：
--
作者：
Hirakawa, Hidehiko;Ishikawa, Suguru;Nagamune, Teruyuki
金黄色葡萄球菌分选酶A（SaSrtA）的催化活性依赖于Ca 2+，因为Ca 2+与活性位点远端的Glu残基的结合稳定了底物结合位点。为了获得Ca 2+非依赖性SaSrtA，我们取代了Ca 2+结合口袋中的两个Glu残基（Glu 105和Glu 108）。虽然单突变降低SaSrtA活性，但Glu 105和Glu 108的突变导致Ca 2+非依赖性活性。动力学分析表明，双突变影响底物结合位点，而不影响底物特异性。这种方法将使我们能够开发适用于各种应用的SaSrtA变体，包括体内位点特异性蛋白质修饰和标记。Biotechnol. Bioeng. 2012; 109：29552961。(C)2012 Wiley Periodicals，Inc.
The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca2+, because binding of Ca2+ to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca2+-independent SaSrtA, we substituted two Glu residues in the Ca2+-binding pocket (Glu105 and Glu108). Although single mutations decreased SaSrtA activity, mutations of both Glu105 and Glu108 resulted in Ca2+-independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site-specific protein modification and labeling. Biotechnol. Bioeng. 2012; 109: 29552961. (C) 2012 Wiley Periodicals, Inc.