Structure and reaction mechanism of basil eugenol synthase

• Published in: PloS one 2007-10, Vol.2 (10), p.e993
• Main Authors: Louie, Gordon V, Baiga, Thomas J, Bowman, Marianne E, Koeduka, Takao, Taylor, John H, Spassova, Snejina M, Pichersky, Eran, Noel, Joseph P
• Format: Article
• Language: English
• Subjects: Acetic acid; Alcohol; Automation; Benzoquinones - chemistry; Binding; Binding Sites; Binding, Competitive; Biochemistry; Biochemistry/Biocatalysis; Biochemistry/Biomacromolecule-Ligand Interactions; Biochemistry/Molecular Evolution; Biophysics/Biocatalysis; Biosynthesis; Biotechnology/Plant Biotechnology; Biotechnology/Small Molecule Chemistry; Bonding; Carbon; Catalysis; Chains; Chemical Biology/Directed Molecular Evolution; Collaboration; Crystallography; Crystallography, X-Ray - methods; Dehydrogenases; Developmental biology; Drosophila; E coli; Enzymes; Escherichia coli; Eugenol; Eugenol - metabolism; Hydrocarbons; Hydrogen; Hydrogen Bonding; Hydrogen bonds; Hydrogen ion concentration; Hydrogen storage; Hydroxides; Inhibitors; Insects; Isoflavones; Isoflavones - chemistry; Lysine; Lysine - chemistry; Models, Chemical; Molecular Conformation; Mutagenesis; NADP; NADP - chemistry; Niacinamide; Nicotinamide; Ocimum basilicum; Ocimum basilicum - enzymology; Oxidoreductases Acting on CH-CH Group Donors - chemistry; Oxidoreductases Acting on CH-CH Group Donors - metabolism; Phenolic compounds; Phenols; Phenols (Class of compounds); Pollinators; Polymerase chain reaction; Protein X; Proteins; Proteomics; Quinone; Quinones; Reaction mechanisms; Reductase; Reductases; Substrate inhibition; Substrates; UDPglucose 4-Epimerase - chemistry; Volatile compounds; X-ray crystallography
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0000993

Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.