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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.


Structure of the GdolS-E248A monomer (left) and dimer(right).The aspartate-rich and NSE metal binding segments are colored redand orange, respectively. The position of the E248A substitution thatpermitted crystallization is indicated as a white band.
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fig2: Structure of the GdolS-E248A monomer (left) and dimer(right).The aspartate-rich and NSE metal binding segments are colored redand orange, respectively. The position of the E248A substitution thatpermitted crystallization is indicated as a white band.

Mentions: The geneencoding GdolS was cloned into a pET-28a vector, which improved thesolution behavior of the expressed protein by decreasing the levelof protein precipitation at the high concentrations required for crystallization.To facilitate crystallization by reducing protein flexibility, theE248A mutation was introduced on the basis of sequence analysis usingthe Surface Entropy Reduction Server (http://services.mbi.ucla.edu/SER/).30 E248 is a surface residue >14Åfrom the nearest active site residue, so its mutation should not significantlyaffect enzyme activity (Figure 2). The E248A mutation was used exclusively for crystallizationpurposes. GdolS-E248A was expressed using the E. coli BL21-CodonPlus(DE3)-RIL strain (Novagen) as described above forwild-type GdolS, except that LB medium containing kanamycin (50 mgL–1) and chloramphenicol (34 mg L–1) was used. Expression was induced with 0.5 mM IPTG at 16 °Cwhen an OD600 of 0.8–1.0 was reached. After expressionfor 18 h, cells were pelleted by centrifugation (3400g for 10 min).


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)

Structure of the GdolS-E248A monomer (left) and dimer(right).The aspartate-rich and NSE metal binding segments are colored redand orange, respectively. The position of the E248A substitution thatpermitted crystallization is indicated as a white band.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Structure of the GdolS-E248A monomer (left) and dimer(right).The aspartate-rich and NSE metal binding segments are colored redand orange, respectively. The position of the E248A substitution thatpermitted crystallization is indicated as a white band.
Mentions: The geneencoding GdolS was cloned into a pET-28a vector, which improved thesolution behavior of the expressed protein by decreasing the levelof protein precipitation at the high concentrations required for crystallization.To facilitate crystallization by reducing protein flexibility, theE248A mutation was introduced on the basis of sequence analysis usingthe Surface Entropy Reduction Server (http://services.mbi.ucla.edu/SER/).30 E248 is a surface residue >14Åfrom the nearest active site residue, so its mutation should not significantlyaffect enzyme activity (Figure 2). The E248A mutation was used exclusively for crystallizationpurposes. GdolS-E248A was expressed using the E. coli BL21-CodonPlus(DE3)-RIL strain (Novagen) as described above forwild-type GdolS, except that LB medium containing kanamycin (50 mgL–1) and chloramphenicol (34 mg L–1) was used. Expression was induced with 0.5 mM IPTG at 16 °Cwhen an OD600 of 0.8–1.0 was reached. After expressionfor 18 h, cells were pelleted by centrifugation (3400g for 10 min).

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.