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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.


Proposed concerted ring closure and allylicelimination of diphosphatein (3R)-NDP. Calculated LUMO of (3R)-nerolidol constrained to the active site volume.
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fig6: Proposed concerted ring closure and allylicelimination of diphosphatein (3R)-NDP. Calculated LUMO of (3R)-nerolidol constrained to the active site volume.

Mentions: The results from incubation of GdolS with NPD (8)are intriguing, because (3R)-NDP appears to be amore efficient substrate than FDP itself. While this may suggest thatthe catalytic mechanism of GdolS proceeds through isomerization ofFDP (1) to NDP prior to 1,10-ring closure, this isomerizationseems unnecessary. Molecular modeling of the LUMO of (3R)-nerolidol (nerolidol was used rather than NDP to simplify the modeling)constrained to the presumed GdolS active site volume shows that inthis conformation the orbitals are aligned to productively drive ringclosure and the allylic elimination of the diphosphate upon promotionof electrons from the HOMO (Figure 6). While product release is generally rate-limitingfor the overall conversion of FDP by terpene synthases, the initialionization of FDP is widely accepted as the rate-determining chemicalstep.51,52 This configuration of NDP is optimal fordirectly generating the transoid-germacryl cationin a concerted manner.


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)

Proposed concerted ring closure and allylicelimination of diphosphatein (3R)-NDP. Calculated LUMO of (3R)-nerolidol constrained to the active site volume.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Proposed concerted ring closure and allylicelimination of diphosphatein (3R)-NDP. Calculated LUMO of (3R)-nerolidol constrained to the active site volume.
Mentions: The results from incubation of GdolS with NPD (8)are intriguing, because (3R)-NDP appears to be amore efficient substrate than FDP itself. While this may suggest thatthe catalytic mechanism of GdolS proceeds through isomerization ofFDP (1) to NDP prior to 1,10-ring closure, this isomerizationseems unnecessary. Molecular modeling of the LUMO of (3R)-nerolidol (nerolidol was used rather than NDP to simplify the modeling)constrained to the presumed GdolS active site volume shows that inthis conformation the orbitals are aligned to productively drive ringclosure and the allylic elimination of the diphosphate upon promotionof electrons from the HOMO (Figure 6). While product release is generally rate-limitingfor the overall conversion of FDP by terpene synthases, the initialionization of FDP is widely accepted as the rate-determining chemicalstep.51,52 This configuration of NDP is optimal fordirectly generating the transoid-germacryl cationin a concerted manner.

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.