VANCOMYCIN BIOSYNTHESIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。对万古霉素类抗生素耐药菌株的出现促使研究人员开发耐药菌株敏感的新型抗生素。一种方法是寻找更有效的强效抗生素的类似物，因为即使抗生素结构的微小变化也会对其活性产生巨大影响。由于技术困难和收率低，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如何在体内合成，其最终目标是通过操纵生物合成中涉及的酶的特异性或顺序来修饰最终产物。本提案研究了这一目标的一个方面，即3,5-二羟基- l -苯基甘氨酸（Dpg）的生物合成途径，Dpg是万古霉素类抗生素中使用的非蛋白质氨基酸。提出了四种酶合成Dpg前体3,5-二羟基苯乙酸酯（Dpa）。本提案主要关注该途径中一种酶DpgC的酶促机制。DpgC是巴豆酶超家族的一员，其特征是能够处理辅酶a （CoA）硫酯。DpgC具有显著的双重催化功能，首先催化双电子氧化生成Dpa-CoA，然后水解CoA酯生成所观察到的酸产物Dpa。氧化步骤特别独特，因为反应的进行显然没有可溶性辅助因子或结合金属离子的参与。如果反应只依赖于分子氧和酶，这将是酶学中一种新的反应机制。酶的氧合，尤其是未活化的亚甲基单位，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。本提案旨在利用x射线晶体结构分析来了解DpgC的机制。对万古霉素生物合成机理的深入了解，可为合理工程或组合生物合成开发新型抗生素提供依据。

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.