Vater J, Stein T, Vollenbroich D, Kruft V, Wittmann-Liebold B, Franke P, Liu L, Zuber P
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Germany.
J Protein Chem. 1997 Jul;16(5):557-64. doi: 10.1023/a:1026386100259.
Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.
来自短短芽孢杆菌的短杆菌肽S合成酶2在其缬氨酸硫醇化中心用硫醇试剂N-[3H]乙基马来酰亚胺进行亲和标记。从酶-抑制剂复合物的胰蛋白酶消化物中，通过两步反相高效液相色谱法以纯形式分离出一个放射性片段。通过液相N端测序结合电喷雾质谱法（ESI-MS）将其鉴定为一个含有硫醇化基序LGG(H/D)S(L/I)的十六肽。通过ESI-MS证明，一个4'-磷酸泛酰巯基乙胺辅因子在其活性丝氨酸处连接到该片段上。这些结果与非核糖体肽生物合成的“多载体模型”一致。已对来自枯草芽孢杆菌的表面活性素合成酶的一些模块的硫醇化、延伸和差向异构化基序进行了位点特异性诱变，以阐明肽生物合成中间步骤中突出的保守氨基酸残基的功能。讨论了多功能肽合成酶的模块化结构。
