6onm

Crystal Structure of (+)-Limonene Synthase Complexed with 8,9-Difluorolinalyl Diphosphate

spinqualitylabelsall models 6onm, resolution 2.70Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6onm is a 1 chain structure with sequence from Citrus sinensis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.7Å Ligands: MN, MWG Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function RLC1_CITSI Catalyzes the conversion of geranyl diphosphate to (+)-(4R)-limonene (PubMed:28272875, PubMed:28272876). Produces exclusively the (+)-enantiomer (PubMed:28272875). Can use neryl diphosphate as substrate (PubMed:28272876). Has no activity with farnesyl diphosphate (PubMed:28272875). Publication Abstract from PubMed Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the alpha-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate. Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.,Morehouse BR, Kumar RP, Matos JO, Yu Q, Bannister A, Malik K, Temme JS, Krauss IJ, Oprian DD ACS Chem Biol. 2019 Sep 20;14(9):2035-2043. doi: 10.1021/acschembio.9b00514. Epub, 2019 Sep 4. PMID:31433159 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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