Institute of Biochemistry, Department of Chemistry and Pharmacy, University of Muenster, Münster, Germany.
Department of Chemistry, Princeton University, Princeton, New Jersey, USA.
Nat Chem Biol. 2017 Sep;13(9):1009-1015. doi: 10.1038/nchembio.2435. Epub 2017 Jul 24.
Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.
非核糖体肽合成酶（NRPSs）是生物活性肽合成的多结构域酶模板。从晶体结构中可以明显看出肽组装过程中的大规模构象变化，但它们的动力学和与催化的偶联仍知之甚少。我们设计了一种 NRPS FRET 传感器，以在溶液中和实时监测来自粘质沙雷氏菌的杆菌肽合成酶 I 的苯丙氨酸结合氨酰化（A）和肽酰载体蛋白结构域之间采用生产性转移构象。配体、底物或中间产物的存在会引起明显的荧光共振能量转移（FRET）读出，这可精确定位到特定构象的群体，或者在两种情况下，定位到构象混合物。焦磷酸开关和赖氨酸电荷传感器控制 A 结构域的结构域交替。苯丙氨酸硫酯和苯丙氨酸-AMP 产物构成了产物抑制和释放的机制，这涉及到有序的装配线肽生物合成。我们的结果代表了从溶液测量中得出的对 NRPS 催化循环构象动力学的见解。
