CRYSTAL STRUCTURE OF THE DICAMBA-DEGRADING RIESKE MONOOXYGENASE FROM PSEUDOMO

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 麦草畏降解菌Rieske单加氧酶的晶体结构 嗜麦芽假单胞菌和其他土壤微生物具有使用三组分麦草畏O-脱甲基酶将除草剂麦草畏（2-甲氧基-3，6-二氯苯甲酸）降解为3，6-二氯水杨酸（DCSA）的能力，所述三组分麦草畏O-脱甲基酶包括铁氧还蛋白、FAD依赖性还原酶和Rieske型单加氧酶。 麦草畏单加氧酶（DMO）与其他Rieske型加氧酶具有适度的序列同一性（约30%），并且除了Rieske铁-硫簇外还含有非血红素单核铁位点。 DMO单氧化麦草畏的甲氧基，而不是芳环。 该反应对于芳香族Rieske加氧酶是非典型的，芳香族Rieske加氧酶通常将其底物的芳香族部分加氧。 因此，DMO通过未知的机制催化不寻常的反应，并且需要结构信息来理解DMO的底物特异性和反应机制。 我们之前已经在Biocars 14 BMC从空间群P31中的DMO晶体收集了2.1 ½原生同步加速器数据集，但是使用各种搜索模型，程序和空间群的分子替换未能提供解决方案。 我们最近在类似于天然晶体的条件下生长了硒代甲硫醚DMO晶体，我们将使用单波长异常衍射获得相。 此外，我们已经在麦草畏的存在下生长了DMO晶体，我们希望确定底物结合复合物的结构。 由于DMO需要还原酶催化完全营业额，我们希望这些晶体含有结合底物或产品。 鉴于我们从天然晶体中获得的高质量衍射，我们预计DMO的Se-met SAD定相和结构解决方案应该是简单的。

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.