Koponen Olli, Tolonen Marja, Qiao Mingqiang, Wahlström Gudrun, Helin Jari, Saris Per E J
Department of Applied Chemistry and Microbiology1 and Institute of Biotechnology2, Viikki Biocentre, University of Helsinki, Finland.
Microbiology (Reading). 2002 Nov;148(Pt 11):3561-3568. doi: 10.1099/00221287-148-11-3561.
Nisin produced by Lactococcus lactis subsp. lactis is a 34-residue antibacterial polypeptide and belongs to a group of post-translationally modified peptides, lantibiotics, with dehydrated residues and cyclic amino acids, lanthionines. These modifications are supposed to be made by enzymes encoded by lanB and lanC genes, found only in biosynthetic operons encoding lantibiotics. To analyse the extent of modification, His-tagged nisin precursors were expressed in nisB and nisC mutant strains. The His-tagged nisin precursors were purified from the cytoplasm of the cells, as lack of NisB or NisC activity impaired translocation of the nisin precursor. The purified His-tagged polypeptides were analysed with trypsin digestion followed by nisin bioassay, SDS-PAGE, N-terminal sequencing and mass spectroscopy. According to the results, nisin precursors from the strain lacking NisB activity were totally unmodified, whereas nisin precursors from the strain lacking NisC activity, but having NisB activity, were dehydrated and devoid of normal lanthionine formation. This is the first experimental evidence showing that NisB is required for dehydration and NisC for correct lanthionine formation in nisin maturation.
乳酸乳球菌乳酸亚种产生的乳链菌肽是一种由34个氨基酸残基组成的抗菌多肽，属于一类翻译后修饰的肽，即羊毛硫抗生素，其含有脱水残基和环状氨基酸（羊毛硫氨酸）。这些修饰被认为是由lanB和lanC基因编码的酶完成的，这些基因仅存在于编码羊毛硫抗生素的生物合成操纵子中。为了分析修饰程度，在nisB和nisC突变菌株中表达了带有His标签的乳链菌肽前体。由于缺乏NisB或NisC活性会损害乳链菌肽前体的转运，因此从细胞的细胞质中纯化了带有His标签的乳链菌肽前体。对纯化的带有His标签的多肽进行胰蛋白酶消化，随后进行乳链菌肽生物测定、SDS-PAGE、N端测序和质谱分析。根据结果，缺乏NisB活性的菌株产生的乳链菌肽前体完全未被修饰，而缺乏NisC活性但具有NisB活性的菌株产生的乳链菌肽前体发生了脱水且没有正常的羊毛硫氨酸形成。这是首个实验证据，表明在乳链菌肽成熟过程中，脱水需要NisB，而正确形成羊毛硫氨酸需要NisC。
