Suliman Muna, Santosh Vishaka, Seegar Tom C M, Dalton Annamarie C, Schultz Kathryn M, Klug Candice S, Barton William A
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, United States of America.
Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
PLoS One. 2017 Aug 31;12(8):e0184271. doi: 10.1371/journal.pone.0184271. eCollection 2017.
The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme's inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.
转肽酶分选酶家族存在于众多革兰氏阳性细菌中，参与多种不同的生理过程，包括将表面蛋白锚定到细胞壁以及菌毛组装。作为必需蛋白，分选酶一直是开发新型抗菌药物的重要研究对象，然而，最近它们作为独特转肽酶的功能已被用于合成新型生物共轭物。然而，出于合成目的，分选酶A（SrtA）介导的共轭反应存在酶本身催化效率较差的问题。因此，为了鉴定具有更高催化效率的SrtA变体，我们采用定向进化来筛选一种催化活性增强的SrtA酶。对改进后的SrtA变体在序列保守性、核磁共振（NMR）和X射线晶体结构以及动力学数据方面的分析表明，存在一种新的催化机制，涉及大的构象变化，将底物传递到活性位点口袋。事实上，使用双电子-电子共振（DEER-EPR）光谱，我们发现，在底物结合后，SrtA会发生类似剪刀的大构象变化，除了重新定位关键催化残基进行酯化反应外，还会同时将分选标签底物转移到活性位点。对分选酶动力学的更好理解将显著增强未来的工程和药物发现工作。
