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首页> 外文期刊>Biochemistry >Site-specific observation of acyl intermediate processing in thiotemplate biosynthesis by fourier transform mass spectrometry: the polyketide module of yersiniabactin synthetase.
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Site-specific observation of acyl intermediate processing in thiotemplate biosynthesis by fourier transform mass spectrometry: the polyketide module of yersiniabactin synthetase.

机译：通过傅里叶变换质谱法对硫代模板生物合成中的酰基中间处理进行定点观察：耶尔西菌素合成酶的聚酮化合物模块。

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摘要

Complex arrays of thioester bound intermediates are present on 100-700 kDa enzymes during the biogenesis of diverse types of pharmacophores and natural product drugs. These multidomain enzymes, known as nonribosomal peptide synthetases and polyketide synthases (NRPSs and PKSs, respectively), synthesize from simple, physiologically available substrates bioactive compounds that can be further tailored by a host of modifying domains (e.g., methylation, cyclization, and epimerization) to increase the complexity of the mature final product. Interrogation of such covalent intermediates using mass spectrometry (MS) presents an underutilized method for understanding the covalent catalysis executed by NRPS and PKS enzymes. For the PKS module (205 kDa) from the yersiniabactin (Ybt) gene cluster of Yersinia pestis, limited proteolysis afforded a key 11 kDa peptide from the acyl-carrier protein (ACP) domain upon which at least five covalent intermediates could be detected (42, 70, 86, 330, and 358 Da). The isotopic resolution achieved by Fourier transform mass spectrometry (FTMS) allowed for the incorporation of substrates with stable isotopes to confirm the structural assignments of three intermediates (86, 330, and 358 Da) on the Ybt biosynthetic pathway to within 1 Da. Approximately 75% of the enzyme capacity is lost to unproductive decarboxylation of malonyl-S-ACP partly constraining the 1.4 min(-)(1) rate of Ybt production in vitro. Acyl transfer to the ACP domain (on the Ybt pathway) was promoted by a factor of approximately 10 over unproductive CO(2) loss in the presence of the cosubstrate S-adenosylmethionine (SAM), with S-adenosylhomocysteine unable to restore the condensation yield observed with SAM. The data are consistent with Claisen condensation from KS to the ACP carrier site being reversible, with the absence of downstream methylation providing more opportunity for unproductive CO(2) loss. Extension of such FTMS-based studies will allow the direct visualization of multiple intermediates in determining the catalytic order of events and kinetics of NRPS and PKS systems.
机译：在各种类型的药效团和天然产物药物的生物合成过程中，与硫酯键合的中间体组成的复杂阵列存在于100-700 kDa的酶上。这些多域酶，分别称为非核糖体肽合成酶和聚酮化合物合酶（分别为NRPS和PKS），是由简单的，可从生理上获得的底物合成的生物活性化合物，这些化合物可以通过许多修饰域（例如，甲基化，环化和差向异构化）进一步定制增加成熟的最终产品的复杂性。使用质谱（MS）询问此类共价中间体为理解NRPS和PKS酶执行的共价催化提供了一种未充分利用的方法。对于鼠疫耶尔森氏菌耶尔西菌素（Ybt）基因簇中的PKS模块（205 kDa），有限的蛋白水解作用从酰基载体蛋白（ACP）域提供了一个关键的11 kDa肽，在其上可以检测到至少五个共价中间体（42 ，70、86、330和358 Da）。通过傅里叶变换质谱（FTMS）获得的同位素分辨率允许掺入具有稳定同位素的底物，从而确认Ybt生物合成途径中的三种中间体（86、330和358 Da）的结构分配在1 Da之内。丙二酰-S-ACP的非生产性脱羧作用损失了大约75％的酶容量，部分限制了体外Ybt生产的1.4分钟（-）（1）速率。在共底物S-腺苷甲硫氨酸（SAM）存在下，酰基转移到ACP域（在Ybt途径上）的活性提高了约10倍，导致非生产性CO（2）损失，S-腺苷同型半胱氨酸无法恢复缩合产率用SAM观察。数据与从KS到ACP载体位点的克莱森缩合是可逆的一致，没有下游甲基化为无生产的CO（2）损失提供了更多的机会。此类基于FTMS的研究的扩展将允许在确定NRPS和PKS系统的事件和动力学的催化顺序时直接可视化多种中间体。

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