荧光假单胞杆菌存在于植物根部，可以产生一系列次生代谢产物，抑制病原微生物对植物根部的侵染，具有良好的生物防治活性，在农业生产上有着非常广泛的应用。2,4-二乙酰基间苯酚（DAPG），是由荧光假单胞杆菌合成的一种具有光谱抗菌活性的抗生素类活性的化合物。参与DAPG生物合成的一些关键基因虽然已经得到生化上的鉴定，但是对于它们的三维结构和催化反应机理目前还并不清楚。本项目计划解析DAPG生物合成的关键酶PhlD，PhlACB及PhlI的晶体结构，并通过定点突变和酶活分析，阐明这些关键酶的催化反应机制。此外，我们还计划利用纯化的蛋白，在溶液中重构DAPG的合成，从而鉴定出DAPG完整的生物合成的过程。这些研究，不但可以加深我们对DAPG生物合成过程和机理的认识，同时也为DAPG合成的酶学改良提供了理论基础。

Pseudomonas fluorescens which colonize in the roots of plants produce a wide variety of secondary metabolites that can effectively prevent the roots being infected by the pathogenic microorganisms. It is therefore a widely used biocontrol agent in agricultural production. The compound 2,4-diacetylphloroglucinol (DAPG) is one of the metabolites produced by pseudomonas fluorescens and displays a remarkably broad spectrum of toxic activity toward bacteria,fungi and even nematodes. Although the DAPG biosynthetic gene cluster has been biochemically characterized, the three dimensional structures and catalytic mechanisms of these key enzymes involved in this process is still unknown. In this proposed project, we plan to solve the crystal structures of the key enzymes involved in the DAPG synthesis, namely PhlD, PhlACB complex and PhlI. Structural analysis in combination with the mutagenesis and enzymatic assay can provide us the detailed catalytic mechanisms of these key enzymes. We also attempt to reconstitute the DAPG biosynthesis in solution in order to identify all the key steps in this process. These studies can not only strengthen our understanding of the key steps and catalytic mechanisms of the DAPG biosynthesis, but pave the road for the improvement for the activity of the enzymes involved in the synthesis as well.