Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92037-0358, USA.
ACS Chem Biol. 2009 Nov 20;4(11):948-57. doi: 10.1021/cb9002128.
A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.
在聚酮合酶（PKS）和非核糖体肽合成酶（NRPS）生物合成途径的基于遗传学的研究与我们对其在活系统中的调控、相互作用和活性的理解之间存在着显著的差距。为了帮助弥合这一差距，我们在这里提出了一种正交活性位点鉴定系统（OASIS），用于 PKS/NRPS 生物合成酶的蛋白质组学鉴定和分析。OASIS 探针针对 PKS/NRPS 活性位点的保守特征，提供模块化合成酶的基于活性的富集，然后通过多维蛋白质鉴定技术（MudPIT）LC-MS/MS 分析进行分析。当应用于模式细菌枯草芽孢杆菌时，这种功能蛋白质组学方法可以检测和定量该生物体中的所有四个模块化合成酶。此外，多个 OASIS 探针的串联应用增强了从复杂蛋白质混合物中鉴定特定 PKS/NRPS 模块的能力。通过扩大 PKS/NRPS 酶的蛋白质组学分析的动态范围，OASIS 为菌株比较、培养条件优化和酶发现提供了一种有价值的工具。
