This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Crystal structure of the Dicamba-degrading Rieske monooxygenase from Pseudomonas maltophilaPseudomonas 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.
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