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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.


Sequence alignment of the terminal helix and loop of GdolS withAT-AS and PR-AS. N299 and S303 in AT-AS and the aligning residuesin GdolS and PR-AS are highlighted in yellow. AT-AS bound to FSDP(farnesyl S-thiolodiphosphate) and [Mg2+]3-PPi,15 with theactive site water colored magenta and interactions with N213, N299,and S303 shown as black dashes (PDB entry 4KUX, chain A). Residues are shown with carbonsin the respective ribbon color, oxygens in red, nitrogens in blue,and hydrogens in white. Phosphorus atoms are colored orange and sulfuratoms yellow; magnesium is shown as green spheres and water as smallred spheres, with the exception of the highlighted magenta water.
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fig4: Sequence alignment of the terminal helix and loop of GdolS withAT-AS and PR-AS. N299 and S303 in AT-AS and the aligning residuesin GdolS and PR-AS are highlighted in yellow. AT-AS bound to FSDP(farnesyl S-thiolodiphosphate) and [Mg2+]3-PPi,15 with theactive site water colored magenta and interactions with N213, N299,and S303 shown as black dashes (PDB entry 4KUX, chain A). Residues are shown with carbonsin the respective ribbon color, oxygens in red, nitrogens in blue,and hydrogens in white. Phosphorus atoms are colored orange and sulfuratoms yellow; magnesium is shown as green spheres and water as smallred spheres, with the exception of the highlighted magenta water.

Mentions: To trace the origin of the oxygenatom of 2, incubations were conducted in buffer containing50% (v/v) H218O and the pentane extractableproducts were analyzed by GC–MS. The mass spectrum of isolatedgermacradien-4-ol showed incorporation of an 18O atom witha 1:0.65 16O:18O ratio (Figure S8), indicating that the hydroxyl group of 2 stems from bulk solvent through quenching of the final carbocationin the reaction sequence. A lack of obvious candidates that mightact as water binding residues to hydrogen bond with and/or activateH2O as a nucleophile led us to examine the structures ofother terpene synthases to gain further insight. A crystallographicstudy of AT-AS revealed a trapped water molecule in the upper activesite hydrogen bonding with N213, N299, and S303 (Figure 4).15 Sequence alignment of AT-AS with GdolS showed that these three residuescorrespond to N218, Y303, and E307 in GdolS. With N218 having beenidentified as influencing the incorporation of water into the GdolSproduct, the function of Y303 and E307 was also investigated by site-directedmutagenesis.


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)

Sequence alignment of the terminal helix and loop of GdolS withAT-AS and PR-AS. N299 and S303 in AT-AS and the aligning residuesin GdolS and PR-AS are highlighted in yellow. AT-AS bound to FSDP(farnesyl S-thiolodiphosphate) and [Mg2+]3-PPi,15 with theactive site water colored magenta and interactions with N213, N299,and S303 shown as black dashes (PDB entry 4KUX, chain A). Residues are shown with carbonsin the respective ribbon color, oxygens in red, nitrogens in blue,and hydrogens in white. Phosphorus atoms are colored orange and sulfuratoms yellow; magnesium is shown as green spheres and water as smallred spheres, with the exception of the highlighted magenta water.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4829482&req=5

fig4: Sequence alignment of the terminal helix and loop of GdolS withAT-AS and PR-AS. N299 and S303 in AT-AS and the aligning residuesin GdolS and PR-AS are highlighted in yellow. AT-AS bound to FSDP(farnesyl S-thiolodiphosphate) and [Mg2+]3-PPi,15 with theactive site water colored magenta and interactions with N213, N299,and S303 shown as black dashes (PDB entry 4KUX, chain A). Residues are shown with carbonsin the respective ribbon color, oxygens in red, nitrogens in blue,and hydrogens in white. Phosphorus atoms are colored orange and sulfuratoms yellow; magnesium is shown as green spheres and water as smallred spheres, with the exception of the highlighted magenta water.
Mentions: To trace the origin of the oxygenatom of 2, incubations were conducted in buffer containing50% (v/v) H218O and the pentane extractableproducts were analyzed by GC–MS. The mass spectrum of isolatedgermacradien-4-ol showed incorporation of an 18O atom witha 1:0.65 16O:18O ratio (Figure S8), indicating that the hydroxyl group of 2 stems from bulk solvent through quenching of the final carbocationin the reaction sequence. A lack of obvious candidates that mightact as water binding residues to hydrogen bond with and/or activateH2O as a nucleophile led us to examine the structures ofother terpene synthases to gain further insight. A crystallographicstudy of AT-AS revealed a trapped water molecule in the upper activesite hydrogen bonding with N213, N299, and S303 (Figure 4).15 Sequence alignment of AT-AS with GdolS showed that these three residuescorrespond to N218, Y303, and E307 in GdolS. With N218 having beenidentified as influencing the incorporation of water into the GdolSproduct, the function of Y303 and E307 was also investigated by site-directedmutagenesis.

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.