Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry
Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry
复制标题
DOI：
10.1021/bi0510781
复制
发表时间：
2005-09-06
期刊：
影响因子：
2.9
通讯作者：
Khosla, C
中科院分区：
文献类型：
--
作者：
Schnarr, NA;Chen, AY;Khosla, C
聚酮化合物天然产物是通过精细蛋白质组装介导的连续链延伸事件生物合成的。在这些系统中蛋白质结合的中间体的简易检测将增加我们对酶的反应性和选择性的理解。我们已经开发了一个串联的蛋白水解/质谱方法监测基板加载和延长6-脱氧腺苷酸B合酶（DEBS），负责生产的大环内酯前体红霉素。关于酮合酶加载和聚酮化合物单元延伸的信息容易获得，而不需要复杂的蛋白质或小分子标记。一组结构相关的底物通过竞争实验和使用LC-MS的动力学测定进行评价，以解析密切相关的物质。观察到酮合酶底物特异性的强立体化学效应。半定量动力学分析允许的决议上的个别酮基肽酶催化的酰基酶形成和聚酮链延伸的步骤的结构和立体化学变化的影响。
Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/ mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.