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Structure and reaction mechanism of basil eugenol synthase.

Louie GV, Baiga TJ, Bowman ME, Koeduka T, Taylor JH, Spassova SM, Pichersky E, Noel JP - PLoS ONE (2007)

Bottom Line: 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 inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction.

View Article: PubMed Central - PubMed

Affiliation: Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, United States of America.

ABSTRACT
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.

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Interactions between EGS and the NADP+ cofactor.Only the EGS polypeptide-chain segments that form direct interactions with the NADP+ cofactor are shown. Hydrogen-bond interactions formed by the cofactor are represented as magenta dashed lines. Atom coloring is the same as in Figure 2A, except that the carbon atoms of the polypeptide-chain segments are green. The blue-colored contours envelope regions greater than 3σ in the NADP-omit electron-density map.
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pone-0000993-g003: Interactions between EGS and the NADP+ cofactor.Only the EGS polypeptide-chain segments that form direct interactions with the NADP+ cofactor are shown. Hydrogen-bond interactions formed by the cofactor are represented as magenta dashed lines. Atom coloring is the same as in Figure 2A, except that the carbon atoms of the polypeptide-chain segments are green. The blue-colored contours envelope regions greater than 3σ in the NADP-omit electron-density map.

Mentions: The structures of EGS complexed with NADP+ or NADPH provide the first structural characterization of nicotinamide-cofactor binding by the PIP family of enzymes (previous crystallographic analyses had yielded only apo-enzyme structures). The cofactor is bound through a large number of polar and non-polar interactions (Figure 3). The adenine ring adopts the anti conformation and is sandwiched between the δ-guanido group of Arg39 and the carboxamide group of Gln87. The adenine-ribose lies in the C3′-endo conformation. The ribose ring is packed against the α-carbons of both Gly14 and Gly17, and the central diphosphate group forms hydrogen bonds with the backbone amide-nitrogens of residues 18 and 19. The protein residues involved in these interactions reside within the Gly14-Xaa-Xaa-Gly17-Xaa-Xaa-Gly20 segment, a canonical sequence-motif for NAD(P) binding [12]. The 2′-phosphate is sequestered by a short loop formed by residues 38–42, and is hydrogen bonded to the side chains of Thr38, Arg39 and Ser42. Thr16 from the glycine-rich loop also hydrogen bonds the 2′-phosphate, as well as the adjacent 3′-hydroxyl group. The nicotinamide-ribose has the C2′-endo conformation, and its hydroxyl groups are involved in hydrogen bonds with residue 111′s carbonyl oxygen, Ser110′s side chain hydroxyl moiety, and the side-chain amino group of Lys132. The nicotinamide ring adopts the anti conformation with its B-face stacked against the side chain of Phe154 and its A-face directed towards the substrate-binding pocket. The nicotinamide carboxamide group forms hydrogen bonds to three backbone atoms (ON7 to 154 N, and NN7 to 112 O and 152 O).


Structure and reaction mechanism of basil eugenol synthase.

Louie GV, Baiga TJ, Bowman ME, Koeduka T, Taylor JH, Spassova SM, Pichersky E, Noel JP - PLoS ONE (2007)

Interactions between EGS and the NADP+ cofactor.Only the EGS polypeptide-chain segments that form direct interactions with the NADP+ cofactor are shown. Hydrogen-bond interactions formed by the cofactor are represented as magenta dashed lines. Atom coloring is the same as in Figure 2A, except that the carbon atoms of the polypeptide-chain segments are green. The blue-colored contours envelope regions greater than 3σ in the NADP-omit electron-density map.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1991597&req=5

pone-0000993-g003: Interactions between EGS and the NADP+ cofactor.Only the EGS polypeptide-chain segments that form direct interactions with the NADP+ cofactor are shown. Hydrogen-bond interactions formed by the cofactor are represented as magenta dashed lines. Atom coloring is the same as in Figure 2A, except that the carbon atoms of the polypeptide-chain segments are green. The blue-colored contours envelope regions greater than 3σ in the NADP-omit electron-density map.
Mentions: The structures of EGS complexed with NADP+ or NADPH provide the first structural characterization of nicotinamide-cofactor binding by the PIP family of enzymes (previous crystallographic analyses had yielded only apo-enzyme structures). The cofactor is bound through a large number of polar and non-polar interactions (Figure 3). The adenine ring adopts the anti conformation and is sandwiched between the δ-guanido group of Arg39 and the carboxamide group of Gln87. The adenine-ribose lies in the C3′-endo conformation. The ribose ring is packed against the α-carbons of both Gly14 and Gly17, and the central diphosphate group forms hydrogen bonds with the backbone amide-nitrogens of residues 18 and 19. The protein residues involved in these interactions reside within the Gly14-Xaa-Xaa-Gly17-Xaa-Xaa-Gly20 segment, a canonical sequence-motif for NAD(P) binding [12]. The 2′-phosphate is sequestered by a short loop formed by residues 38–42, and is hydrogen bonded to the side chains of Thr38, Arg39 and Ser42. Thr16 from the glycine-rich loop also hydrogen bonds the 2′-phosphate, as well as the adjacent 3′-hydroxyl group. The nicotinamide-ribose has the C2′-endo conformation, and its hydroxyl groups are involved in hydrogen bonds with residue 111′s carbonyl oxygen, Ser110′s side chain hydroxyl moiety, and the side-chain amino group of Lys132. The nicotinamide ring adopts the anti conformation with its B-face stacked against the side chain of Phe154 and its A-face directed towards the substrate-binding pocket. The nicotinamide carboxamide group forms hydrogen bonds to three backbone atoms (ON7 to 154 N, and NN7 to 112 O and 152 O).

Bottom Line: 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 inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction.

View Article: PubMed Central - PubMed

Affiliation: Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, United States of America.

ABSTRACT
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.

Show MeSH