Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics
Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics
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DOI：
10.1002/pro.3138
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发表时间：
2017-03
期刊：
影响因子：
8
通讯作者：
S. Paul;H. Ishida;L. T. Nguyen;Zhihong Liu;H. Vogel
中科院分区：
文献类型：
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作者：
S. Paul;H. Ishida;L. T. Nguyen;Zhihong Liu;H. Vogel
弗留利霉素是由弗留利游动放线菌产生的环状脂十肽抗生素。与相关脂肽药物达托霉素类似，弗留利霉素的肽骨架由大型多酶非核糖体肽合成酶（NRPS）系统合成。LipD蛋白在弗留利霉素的酰化反应中起主要作用。脂肪酸基团的连接促进其抗生素活性。系统发育分析表明，LipD是最密切相关的其他独立的酰基载体蛋白（ACP），其基因位于附近的NRPS基因簇。在这里，我们报告了apo-LipD的溶液NMR结构与来自脂肪酸合酶（FAS）、聚酮合酶和NRPS系统的其他四螺旋束形成ACP非常相似。通过记录NMR动力学数据，我们发现由于磷酸泛酰巯基乙胺部分的连接，holo-LipD中的主链运动比apo-LipD中的更受限制。在差示扫描量热法实验中也观察到这种增强的holo-LipD稳定性。此外，我们证明，与其他几种ACP不同，LipD的折叠不依赖于二价阳离子的存在，尽管Mg 2+或Ca 2+的存在可以增加蛋白质的稳定性。我们提出，导致稳定性增强的holo-LipD三级结构中的小的结构重排对于酰化反应的同源酶识别是重要的。我们的结果还突出了LipD和FAS-ACP与A.弗留利酵母中，这将允许酰基辅酶A连接酶优先与LipD相互作用，而不是结合FAS-ACP。
Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo‐LipD is very similar to other four‐helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo‐LipD are more restricted than in apo‐LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo‐LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg2+ or Ca2+ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo‐LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS‐ACP from A. friuliensis that would allow the acyl‐CoA ligase to interact preferentially with the LipD instead of binding to the FAS‐ACP.