Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase
Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase
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DOI：
10.1021/bi026592a
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发表时间：
2002-11-12
期刊：
影响因子：
2.9
通讯作者：
Sieber, SA
中科院分区：
文献类型：
--
作者：
Tseng, CC;Bruner, SD;Sieber, SA
当提供可溶性β-羟基-丁酰-七肽基硫酯底物时，产生脂肽表面活性素（Srf TE）的非核糖体肽合成酶的C-末端硫酯酶结构域保留产生表面活性素的环状肽内酯骨架的自主能力。利用最近解决的晶体结构[Bruner，S. D、等人（2002）Structure 10，301-310]，活性位点亲核体Ser 8 O变为Cys，催化三联体的其他成员Asp 107和His 207变为Ala，所得突变体缺乏可检测的活性。活性位点中的两个阳离子侧链，Lys 111和Arg 120，被改变为Ala，导致产物水解的分配增加，如P26 G突变体，模仿脂肪酶的行为。为了评估Srf TE使用的底物中的识别元素，对脂肪酰基、七肽和硫酯离去基团进行了改变，并对所得底物进行了环化或水解的动力学能力和产物通量的表征。在底物的所有三个部分中鉴定了可接受的环化改变，尽管变化的容许限度不同。此外，解决了Srf TE与二肽基硼酸酯抑制剂的共晶结构，说明了底物的关键结合决定簇。在结构和生化数据的基础上，将表面活性肽的环化构象模拟为酶的活性位点。
The C-terminal thioesterase domain of the nonribosomal peptide synthetase producing the lipopetide surfactin (Srf TE) retains autonomous ability to generate the cyclic peptidolactone skeleton of surfactin when provided with a soluble beta-hydroxy-butyryl-heptapeptidyl thioester substrate. Utilizing the recently solved crystal structure [Bruner, S. D., et al. (2002) Structure 10, 301-310], the active-site nucleophile, Ser8O, was changed to Cys, and the other members of the catalytic triad, Asp107 and His207, were changed to Ala, with the resulting mutants lacking detectable activity. Two cationic side chains in the active site, Lys111 and Arg120, were changed to Ala, causing an increased partitioning of the product to hydrolysis, as did a P26G mutant, mimicking the behavior of lipases. To evaluate recognition elements in substrates used by Srf TE, alterations to the fatty acyl group, the heptapeptide, and the thioester leaving group were made, and the resulting substrates were characterized for kinetic competency and flux of product to cyclization or hydrolysis. Alterations that could be accepted for cyclization were identified in all three parts of the substrate, although tolerance limits for changes varied. In addition, cocrystal structures of Srf TE with dipeptidyl boronate inhibitors were solved, illustrating the critical binding determinants of the substrate. On the basis of the structures and biochemical data, the cyclizing conformation of the surfactin peptide was modeled into the enzyme active site.