6ONM

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

Summary for 6ONM

• Entry DOI: 10.2210/pdb6onm/pdb
• Descriptor: (+)-limonene synthase, MANGANESE (II) ION, (3R)-8-fluoro-7-(fluoromethyl)-3-methylocta-1,6-dien-3-yl trihydrogen diphosphate, ... (4 entities in total)
• Functional Keywords: terpene synthase, enantiomer, terpene synthase fold, monoterpene, fluoro analog, metal binding, lyase
• Biological source: Citrus sinensis (Sweet orange)
• Total number of polymer chains: 1
• Total formula weight: 70956.22

Authors

Prem Kumar, R.,Morehouse, B.R.,Yu, Q.,Oprian, D.D. (deposition date: 2019-04-22, release date: 2019-09-04, Last modification date: 2023-10-11)

Primary citation

Morehouse, B.R.,Kumar, R.P.,Matos, J.O.,Yu, Q.,Bannister, A.,Malik, K.,Temme, J.S.,Krauss, I.J.,Oprian, D.D.
Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.
Acs Chem.Biol., 14:2035-2043, 2019

PubMed Abstract: 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 -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 α-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.

PubMed: 31433159
DOI: 10.1021/acschembio.9b00514

Experimental method

X-RAY DIFFRACTION (2.7 Å)