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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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Structural comparison of EGS and UDP-galactose epimerase.(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (color coding as shown in inset). For clarity, only the NADP+ cofactor of EGS is shown. (B) Comparison of NAD(P)-cofactor conformation and substrate-analog binding in EGS and UDP-galactose epimerase. The binding of the EGS competitive inhibitor (7S,8S)-ethyl (7,8-methylene)-dihydroferulate (EMDF) is described in detail in the text and Figure 5. The coloring of the polypeptide-chain segments is the same as in (A). The inset shows the coloring used for the carbon atoms of the nicotinamide cofactors, EMDF bound to EGS, and UDP-glucose bound to UDP-galactose-4-epimerase.
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pone-0000993-g004: Structural comparison of EGS and UDP-galactose epimerase.(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (color coding as shown in inset). For clarity, only the NADP+ cofactor of EGS is shown. (B) Comparison of NAD(P)-cofactor conformation and substrate-analog binding in EGS and UDP-galactose epimerase. The binding of the EGS competitive inhibitor (7S,8S)-ethyl (7,8-methylene)-dihydroferulate (EMDF) is described in detail in the text and Figure 5. The coloring of the polypeptide-chain segments is the same as in (A). The inset shows the coloring used for the carbon atoms of the nicotinamide cofactors, EMDF bound to EGS, and UDP-glucose bound to UDP-galactose-4-epimerase.

Mentions: The PIP-family enzymes belong to a larger superfamily of NAD(P)-dependent dehydrogenases, the short-chain dehydrogenases/reductases (SDRs) [13]. The most similar member of the larger superfamily is UDP-galactose 4-epimerase [14] (PDB entry 1KVQ), which provided a template for the binding modes of both the nicotinamide cofactor and substrate in the earlier structural analyses of the apo-forms of IFR, PCBER and PLR [6], [7]. Indeed, UDP-galactose 4-epimerase possesses a C-terminal domain that is similar topologically to the C-terminal domains of the PIP-family proteins (Figure 4A). In the UDP-galactose 4-epimerase crystal structure, a cavity within the C-terminal domain is positioned next to the nicotinamide ring of the NAD+ cofactor and is occupied by the substrate, UDP galactose. The corresponding cavity in EGS is much smaller in volume and the side chains lining the cavity are more non-polar in character. These properties of the EGS substrate-binding pocket are consistent with the smaller size and greater hydrophobicity of the EGS substrate, the acetate ester of coniferyl alcohol. Notably, the conformation of NAD+ bound to UDP-galactose 4-epimerase differs markedly from that of NADP+ bound to EGS, particularly in the conformation of the nicotinamide ring (Figure 4B). In the UDP-galactose 4-epimerase/NAD+ complex, the nicotinamide ring adopts the syn conformation, consistent with the class-B oxidoreductase activity of the enzyme. In contrast, the anti-conformer of the nicotinamide ring observed in EGS is consistent with the class-A reductase (donation of the pro-R hydride) activity of the PIP-family enzymes. The orientation of the nicotinamide ring in EGS appears to be influenced largely by interactions of the carboxamide group with the polypeptide-chain backbone, an observation also made previously for the SDRs [15]. Also, EGS possesses an additional loop (residues 38–42) that forms a binding pocket for the 2′-phosphate group of NADP(H). This loop is absent in UDP-galactose 4-epimerase, but occurs with variable length in all PIP-family proteins.


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)

Structural comparison of EGS and UDP-galactose epimerase.(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (color coding as shown in inset). For clarity, only the NADP+ cofactor of EGS is shown. (B) Comparison of NAD(P)-cofactor conformation and substrate-analog binding in EGS and UDP-galactose epimerase. The binding of the EGS competitive inhibitor (7S,8S)-ethyl (7,8-methylene)-dihydroferulate (EMDF) is described in detail in the text and Figure 5. The coloring of the polypeptide-chain segments is the same as in (A). The inset shows the coloring used for the carbon atoms of the nicotinamide cofactors, EMDF bound to EGS, and UDP-glucose bound to UDP-galactose-4-epimerase.
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Related In: Results  -  Collection

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pone-0000993-g004: Structural comparison of EGS and UDP-galactose epimerase.(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (color coding as shown in inset). For clarity, only the NADP+ cofactor of EGS is shown. (B) Comparison of NAD(P)-cofactor conformation and substrate-analog binding in EGS and UDP-galactose epimerase. The binding of the EGS competitive inhibitor (7S,8S)-ethyl (7,8-methylene)-dihydroferulate (EMDF) is described in detail in the text and Figure 5. The coloring of the polypeptide-chain segments is the same as in (A). The inset shows the coloring used for the carbon atoms of the nicotinamide cofactors, EMDF bound to EGS, and UDP-glucose bound to UDP-galactose-4-epimerase.
Mentions: The PIP-family enzymes belong to a larger superfamily of NAD(P)-dependent dehydrogenases, the short-chain dehydrogenases/reductases (SDRs) [13]. The most similar member of the larger superfamily is UDP-galactose 4-epimerase [14] (PDB entry 1KVQ), which provided a template for the binding modes of both the nicotinamide cofactor and substrate in the earlier structural analyses of the apo-forms of IFR, PCBER and PLR [6], [7]. Indeed, UDP-galactose 4-epimerase possesses a C-terminal domain that is similar topologically to the C-terminal domains of the PIP-family proteins (Figure 4A). In the UDP-galactose 4-epimerase crystal structure, a cavity within the C-terminal domain is positioned next to the nicotinamide ring of the NAD+ cofactor and is occupied by the substrate, UDP galactose. The corresponding cavity in EGS is much smaller in volume and the side chains lining the cavity are more non-polar in character. These properties of the EGS substrate-binding pocket are consistent with the smaller size and greater hydrophobicity of the EGS substrate, the acetate ester of coniferyl alcohol. Notably, the conformation of NAD+ bound to UDP-galactose 4-epimerase differs markedly from that of NADP+ bound to EGS, particularly in the conformation of the nicotinamide ring (Figure 4B). In the UDP-galactose 4-epimerase/NAD+ complex, the nicotinamide ring adopts the syn conformation, consistent with the class-B oxidoreductase activity of the enzyme. In contrast, the anti-conformer of the nicotinamide ring observed in EGS is consistent with the class-A reductase (donation of the pro-R hydride) activity of the PIP-family enzymes. The orientation of the nicotinamide ring in EGS appears to be influenced largely by interactions of the carboxamide group with the polypeptide-chain backbone, an observation also made previously for the SDRs [15]. Also, EGS possesses an additional loop (residues 38–42) that forms a binding pocket for the 2′-phosphate group of NADP(H). This loop is absent in UDP-galactose 4-epimerase, but occurs with variable length in all PIP-family proteins.

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