6onm
From Proteopedia
Crystal Structure of (+)-Limonene Synthase Complexed with 8,9-Difluorolinalyl Diphosphate
Structural highlights
FunctionRLC1_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).[1] [2] Publication Abstract from PubMedLinalyl 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[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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