Stein T, Vater J, Kruft V, Otto A, Wittmann-Liebold B, Franke P, Panico M, McDowell R, Morris H R
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin-Charlottenburg, Germany.
J Biol Chem. 1996 Jun 28;271(26):15428-35. doi: 10.1074/jbc.271.26.15428.
Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.
短杆菌肽S合成酶1和2在其硫醇化中心进行亲和标记，方法是与氨基酸底物进行硫酯化反应，或者与硫醇试剂N-乙基马来酰亚胺结合底物保护技术进行特异性烷基化。标记的蛋白质通过溴化氰化学消化或蛋白酶消化。开发并使用了一种高效的多步高压液相色谱方法，以纯形式分离短杆菌肽S合成酶所有五个硫醇化中心的活性位点肽片段。通过N端测序、质谱和氨基酸分析研究这些片段的结构。每个活性位点肽片段都包含共有基序LGG(H/D)S(L/I)，这在非核糖体肽生物合成中对硫酯形成具有特异性。结果表明，在短杆菌肽S合成酶多酶系统的每个氨基酸活化模块中，一个4'-磷酸泛酰巯基乙胺辅因子连接到硫醇化基序的中心丝氨酸上，形成氨基酸底物的硫酯结合位点并催化延伸过程。我们的数据有力支持了多功能模板上非核糖体肽生物合成的“多载体模型”，对此进行了详细讨论。
