Influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by Lactococcus lactis.
Influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by Lactococcus lactis.
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乳链菌肽前导肽中的氨基酸取代对乳酸乳球菌乳链菌肽生物合成和分泌的影响。
DOI：
10.1016/s0021-9258(17)41899-0
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
W. Vos
中科院分区：
文献类型：
--
作者：
J. R. V. D. Meer;H. Rollema;R. Siezen;M. Beerthuyzen;O. Kuipers;W. Vos
含羊毛硫氨酸的小抗微生物肽（称为羊毛硫抗生素）的结构基因编码N-末端前导序列，其不存在于成熟肽中，但在成熟过程的某个阶段被切割掉。不同羊毛硫抗生素的前导序列共享许多相同的氨基酸残基，但它们明显不同于sec依赖的蛋白质输出信号序列。我们研究了羊毛硫抗生素乳酸链球菌素的前导序列的作用，这是由乳酸乳球菌产生和分泌，通过创建定点突变的前导肽序列中的各个位置。在Arg-1和Ala-4，但不是在保守的Pro-2的突变，强烈影响的前导序列的加工，并导致在细胞外积累的生物活性的前体肽。氨基酸分析和1H NMR研究表明，Ala-4->Asp突变的前体肽含有一个修饰的nisin结构部分，（突变的）未修饰的前导序列仍然连接在它上面。这些结果证实前导肽的切割是乳链菌肽成熟的最后一步，并且是产生生物活性肽所必需的。前导肽中的几个突变，即Pro-2->Gly、Pro-2->瓦尔、Asp-7->Ala、Lys-9->Leu、Ser-10->Ala/Ser-12->Ala和瓦尔-11->Asp/瓦尔-13->Glu，对乳链菌肽的产生和分泌没有任何可检测的影响，尽管其中一些突变影响高度保守的残基。当在乳链菌肽前导肽的-18至-15区域中产生突变（即Phe-18->Leu，Leu-16->Lys，Asp-15->Ala）时，不能检测到乳链菌肽或其前体的分泌或细胞内积累。这表明这些保守残基参与成熟过程，并可能与lactic-specific修饰酶相互作用。
Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence. Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4–>Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it. The Ala-4–>Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide. Several mutations, i.e. Pro-2–>Gly,Pro-2–>Val, Asp-7–>Ala,Lys-9–>Leu,Ser-10–>Ala/Ser-12–>Ala and Val-11–>Asp/Val-13–>Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues. When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e. Phe-18–>Leu,Leu-16–>Lys,Asp-15–>Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.