CRYSTAL STRUCTURE OF THE DICAMBA-DEGRADING RIESKE MONOOXYGENASE FROM PSEUDOMO

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 麦芽假单胞菌降解麦芽威的Rieske单加氧酶的晶体结构 麦芽假单胞菌和其他土壤微生物能够利用由铁氧还蛋白、FAD依赖的还原酶和Rieske类型的单加氧酶组成的三组分Dicamba O-去甲基酶将除草剂Dicamba(2-甲氧基-3，6-二氯苯甲酸)降解为3，6-二氯水杨酸(DCSA)。Dicamba单加氧酶(DMO)与其他Rieske类型的加氧酶有适度的序列同源性(约30%)，除了Rieske铁-硫簇外，还含有一个非血红素单核铁位点。DMO单氧化麦草畏的甲氧基，而不是芳香环。这个反应对于芳香族Rieske加氧酶来说是非典型的，它通常氧化其底物的芳香族部分。因此，DMO以未知的机制催化一种不寻常的反应，需要结构信息来了解DMO的底物专一性和反应机理。 我们之前已经在Biocars 14BMC上从P31空间群中的DMO晶体收集了2.1°天然同步加速器数据集，然而使用各种搜索模型、程序和空间群进行分子替换未能提供解决方案。我们最近在与天然晶体相似的条件下生长了硒蛋氨酸DMO晶体，并将利用单波长反常衍射获得物相。此外，我们还在麦草畏存在下生长了DMO晶体，我们希望确定底物结合络合物的结构。由于DMO需要还原酶来催化完全周转，我们希望这些晶体含有结合的底物或产物。考虑到我们从天然晶体获得的高质量的衍射，我们预计对于DMO的Se-Met SAD相变和结构解决方案应该是直接的。

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. Crystal structure of the Dicamba-degrading Rieske monooxygenase from Pseudomonas maltophila Pseudomonas maltophila and other soil microbes have the ability to degrade the herbicide Dicamba (2-methoxy-3,6-dichlorobenzoic acid) to 3,6-dichlorosalicylic acid (DCSA) using a three-component dicamba O-demethylase that comprises a ferredoxin, a FAD-dependent reductase, and a Rieske-type monooxygenase. The dicamba monooxygenase (DMO) has modest sequence identity (approximately 30%) to other Rieske-type oxygenases and contains a non-heme mononuclear iron site in addition to the Rieske iron-sulfur cluster. DMO monooxygenates the methoxy group, rather than the aromatic ring, of dicamba. This reaction is atypical for aromatic Rieske oxygenases, which typically oxygenate the aromatic moiety of their substrates. Therefore, DMO catalyzes an unusual reaction by an unknown mechanism and structural information is required to understand both the substrate specificity and reaction mechanism of DMO. We have previously collected a 2.1 ¿ native synchrotron dataset at Biocars 14BMC from a crystal of DMO in space group P31, however molecular replacement using a variety of search models, programs, and space groups has failed to deliver a solution. We have recently grown crystals of selenomethionine DMO in conditions similar to the native crystals, and we will obtain phases using single wavelength anomalous diffraction. In addition, we have grown crystals of DMO in the presence of dicamba, and we hope to determine the structure of the substrate-bound complex. Because DMO requires a reductase to catalyze complete turnover, we are hopeful that these crystals contain either bound substrate or product. Given the high-quality diffraction that we have obtained from native crystals, we expect that Se-met SAD phasing and structure solution for DMO should be straightforward.