Structure and reaction mechanism of basil eugenol synthase.
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.
Louie
Gordon V
GV
Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, United States of America.
Baiga
Thomas J
TJ
Bowman
Marianne E
ME
Koeduka
Takao
T
Taylor
John H
JH
Spassova
Snejina M
SM
Pichersky
Eran
E
Noel
Joseph P
JP
D016227:Benzoquinones / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D005054:Eugenol / Q000378:metabolism*; D006860:Hydrogen Bonding; D007529:Isoflavones / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008968:Molecular Conformation; D009249:NADP / Q000737:chemistry; D018649:Ocimum basilicum / Q000201:enzymology*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism; D014534:UDPglucose 4-Epimerase / Q000737:chemistry
D016227:Benzoquinones; D007529:Isoflavones; C004532:quinone; D005054:Eugenol; D009249:NADP; D044925:Oxidoreductases Acting on CH-CH Group Donors; C529970:eugenol synthase, Ocimum basilium; D014534:UDPglucose 4-Epimerase; D008239:Lysine