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Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

Grundy DJ, Chen M, González V, Leoni S, Miller DJ, Christianson DW, Allemann RK - Biochemistry (2016)

Bottom Line: Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture.The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled.Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, Cardiff University , Park Place, Cardiff CF10 3AT, United Kingdom.

ABSTRACT
The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent.

No MeSH data available.


Possible mechanismfor the germacradien-4-ol synthase (GdolS)-catalyzedconversion of farnesyl diphosphate (1) to (−)-germaradien-4-ol(2).
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fig1: Possible mechanismfor the germacradien-4-ol synthase (GdolS)-catalyzedconversion of farnesyl diphosphate (1) to (−)-germaradien-4-ol(2).

Mentions: Class I sesquiterpene synthases share a common α-helicalstructure with the active site located in a hydrophobic cleft betweentwo helices containing the highly conserved metal binding motifs DDXXD/Eand NSE/DTE.9 Prior to initiation of thecyclization cascade through Mg2+-dependent diphosphatecleavage, the active site closes to protect the carbocationic intermediatesfrom premature quenching by bulk solvent molecules.10,11 The enclosed active site volume of a terpenoid cyclase is typicallyjust slightly larger than that of the substrate, which ensures a snugfit between the enzyme and the flexible isoprenoid substrate.12,13 However, X-ray crystallographic studies show that sometimes a watermolecule can be trapped in the closed conformation of a terpenoidcyclase active site along with a bound substrate analogue or product.14,15 Notably, a limited number of the sesquiterpene synthases produceterpenoid alcohols and epoxides containing a single oxygen atom, presumablyderived from a water molecule bound in the terpene cyclase activesite along with the isoprenoid diphosphate substrate.16−19 The bacterial sesquiterpene synthase (−)-germacradien-4-olsynthase (GdolS) converts farnesyl diphosphate (FDP, 1) into the macrocyclic terpene alcohol (−)-germacradien-4-ol(2) (Figure 1). The enzyme exerts significant control over a catalyticwater molecule, which quenches the final carbocation intermediateof the reaction cascade with high stereospecificity and regiospecificity.20


Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

Grundy DJ, Chen M, González V, Leoni S, Miller DJ, Christianson DW, Allemann RK - Biochemistry (2016)

Possible mechanismfor the germacradien-4-ol synthase (GdolS)-catalyzedconversion of farnesyl diphosphate (1) to (−)-germaradien-4-ol(2).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829482&req=5

fig1: Possible mechanismfor the germacradien-4-ol synthase (GdolS)-catalyzedconversion of farnesyl diphosphate (1) to (−)-germaradien-4-ol(2).
Mentions: Class I sesquiterpene synthases share a common α-helicalstructure with the active site located in a hydrophobic cleft betweentwo helices containing the highly conserved metal binding motifs DDXXD/Eand NSE/DTE.9 Prior to initiation of thecyclization cascade through Mg2+-dependent diphosphatecleavage, the active site closes to protect the carbocationic intermediatesfrom premature quenching by bulk solvent molecules.10,11 The enclosed active site volume of a terpenoid cyclase is typicallyjust slightly larger than that of the substrate, which ensures a snugfit between the enzyme and the flexible isoprenoid substrate.12,13 However, X-ray crystallographic studies show that sometimes a watermolecule can be trapped in the closed conformation of a terpenoidcyclase active site along with a bound substrate analogue or product.14,15 Notably, a limited number of the sesquiterpene synthases produceterpenoid alcohols and epoxides containing a single oxygen atom, presumablyderived from a water molecule bound in the terpene cyclase activesite along with the isoprenoid diphosphate substrate.16−19 The bacterial sesquiterpene synthase (−)-germacradien-4-olsynthase (GdolS) converts farnesyl diphosphate (FDP, 1) into the macrocyclic terpene alcohol (−)-germacradien-4-ol(2) (Figure 1). The enzyme exerts significant control over a catalyticwater molecule, which quenches the final carbocation intermediateof the reaction cascade with high stereospecificity and regiospecificity.20

Bottom Line: Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture.The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled.Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, Cardiff University , Park Place, Cardiff CF10 3AT, United Kingdom.

ABSTRACT
The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent.

No MeSH data available.