VANCOMYCIN BIOSYNTHESIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。对万古霉素类抗生素具有耐药性的细菌菌株的出现，促使研究人员开发对耐药菌株敏感的新型抗生素。一种方法是寻找更有效的抗生素的类似物，因为即使抗生素结构的微小变化也会对它们的活性产生巨大影响。由于技术困难和产率低，化学合成这类类似物在很大程度上是不切实际的。另一种选择是研究这些抗生素是如何在体内合成的，最终目标是通过操纵参与生物合成的酶的特异性或顺序来修饰最终产品。这个方案研究了这一目标的一个方面，即3，5-二羟基-L-苯甘氨酸的生物合成途径，这是一种用于万古霉素类抗生素的非蛋白生成氨基酸。提出了合成DPG前体3，5-二羟基苯乙酸酯(DPA)的四种酶。这一建议侧重于该途径中的一种酶DpgC的酶机制。DpgC是巴豆酶超家族中的一员，其特征是具有处理辅酶A(CoA)硫代酯的能力。DpgC具有显著的双重催化作用，首先催化双电子氧化生成DPA-CoA，然后将CoA酯水解形成酸产物DPA。氧化步骤特别独特，因为反应显然是在没有可溶性辅因子或结合金属离子参与的情况下进行的。如果该反应仅依赖于分子氧和酶，这将代表一种新的酶学机制。酶的氧化，特别是未活化的亚甲基单元，总是使用辅助因子，如黄素和血红素和/或还原活性金属，如铁或铜。本研究试图通过X射线晶体结构分析来了解DpgC的形成机理。对万古霉素生物合成机理的详细了解可用于通过合理的工程或组合生物合成来开发新的抗生素。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The emergence of bacterial strains resistant to vancomycin group antibiotics has driven researchers to develop novel antibiotics to which resistant strains are susceptible. One approach is to pursue analogs of potent antibiotics that have even greater efficacy, as even slight changes to antibiotic structure can have dramatic effects on their activity. Chemical synthesis of such analogs is largely impractical, due to the technical difficulty and low yield. An alternative centers on the study of how these antibiotics are synthesized in vivo, with the ultimate goal of modifying the end product through the manipulation of the specificities or order of the enzymes involved in the biosynthesis. This proposal studies one aspect of this goal, the biosynthetic pathway of 3,5-dihydroxy-L-phenylglycine (Dpg), a non-proteinogenic amino acid used in vancomycin group antibiotics. Four enzymes are proposed to synthesize 3,5-dihydroxyphenylacetate (Dpa), a precursor to Dpg. This proposal focuses on the enzymatic mechanism of one enzyme in the pathway, DpgC. DpgC is a member of the crotonase superfamily of proteins characterized by the ability to process Coenzyme A (CoA) thioesters. DpgC posseses a remarkable dual function in catalysis, first catalyzing the two electron oxidation to Dpa-CoA, then hydrolyzing the CoA esters to form the observed acid product, Dpa. The oxidation step is particularly unique, in that the reaction apparently proceeds without the participation of soluble cofactors or bound metal ions. If the reaction is dependant only on molecular oxygen and the enzyme it would represent a novel mechanism in enzymology. Enzyme oxygenations, especially of unactivated methylene units, invariably use cofactors such as flavins and hemes and/or redux-active metals such as iron or copper. This proposal seeks to understand the mechanism of DpgC using X-ray crystallographic structural analysis. A detailed understanding of the mechanism of vancomycin biosynthesis can be applied toward the development on novel antibiotics through rational engineering or combinatorial biosynthesis.