Determining sites of interaction between prenisin and its modification enzymes NisB and NisC
Determining sites of interaction between prenisin and its modification enzymes NisB and NisC
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DOI：
10.1111/j.1365-2958.2011.07846.x
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发表时间：
2011-11-01
影响因子：
3.6
通讯作者：
Kuipers, Oscar P.
中科院分区：
文献类型：
--
作者：
Khusainov, Rustem;Heils, Rene;Kuipers, Oscar P.
虽然乳链菌肽是一个模型lantibiotic，我们的知识的具体相互作用prenisin与其修饰酶仍然零碎。在这里，我们证明了乳链菌肽修饰酶NisB和NisC可以在体外从乳酸乳球菌中通过工程化的His标记的prenisin被拉下来。这种方法使我们能够确定重要的prenisin分子间的相互作用，其修饰机制在L。乳酸菌。我们证明了（i）NisB与前体乳链菌肽的相互作用比NisC更强，（ii）保持乳链菌肽前导序列完整的前肽部分的缺失导致缺乏结合，（iii）高度保守残基W 616、F342 A、Y346 F和P639 A的NisB点突变体仍然能够与乳链菌肽结合，（iv）NisB Delta（77-79）Y80 F突变体降低了NisB-前霉素相互作用的水平，并导致未修饰的前霉素，（v）NisC中活性位点残基H331 A的取代导致更高量的共纯化复合物，（vi）NisB以二聚体的形式存在，和（vii）前导序列的FNLD（-18至-15）区域是不仅与NisB结合而且与NisC结合的重要位点。
Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Delta(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.