Chakicherla A, Hansen J N
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
J Biol Chem. 1995 Oct 6;270(40):23533-9. doi: 10.1074/jbc.270.40.23533.
Biosynthesis of lantibiotics such as nisin and subtilin involves post-translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium. We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene. The chimeras consisted of a normal subtilin leader region (SL), fused to nisin-subtilin chimeric structural regions, one of which was SL-Nis1-11-Sub12-32, in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin. This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity. A succinylated form of the chimera was also produced. Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (SL-Sub1-11-Nis12-34). It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself. These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide.
诸如乳链菌肽和枯草菌素等羊毛硫抗生素的生物合成涉及翻译后修饰，包括丝氨酸和苏氨酸的脱水、硫醚交联的形成、转运、前导序列的切割以及释放到培养基中。我们通过在枯草芽孢杆菌168菌株中构建并表达乳链菌肽 - 枯草菌素嵌合前肽，研究了进行这些修饰的细胞机制。该菌株除了前枯草菌素基因外，拥有合成枯草菌素的所有细胞机制。嵌合体由正常的枯草菌素前导区（SL）与乳链菌肽 - 枯草菌素嵌合结构区融合而成，其中之一是SL - Nis1 - 11 - Sub12 - 32，其结构区的N端部分源自乳链菌肽，C端部分源自枯草菌素。通过反相高效液相色谱图谱、质子核磁共振光谱、质谱分析和生物活性确定，该嵌合体被准确且高效地转化为相应的成熟羊毛硫抗生素。还产生了该嵌合体的琥珀酰化形式。另一种嵌合体则相反，结构区的N端是枯草菌素序列，C端是乳链菌肽序列（SL - Sub1 - 11 - Nis12 - 34）。它被加工成产物的异质混合物，其中没有一种具有正确加工多肽的特征，但确实含有一种有活性的次要成分，其比活性大大超过乳链菌肽本身。这些结果与早期发表的结果一起表明，加工需要前羊毛硫抗生素肽与加工机制之间的特异性识别，并且为了使加工正确发生，前肽的前导区N端部分和结构区C端部分必须有适当的组合。
