Biochemistry. Apr/11/2016; 55(14): 2112-2121

Published online Mar/20/2016

PMID: 26998816

PMC: 4829482

doi: 10.1021/acs.biochem.6b00115

Mechanism of Germacradien-4-ol Synthase-ControlledWater Capture

David W. Christianson

Rudolf K. Allemann

Abstract

The sesquiterpene synthase germacradiene-4-olsynthase (GdolS)from Streptomyces citricolor is one of only a fewknown high-fidelity terpene synthases that convert farnesyl diphosphate(FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248Adiffracted to 1.50 Å and revealed a typical class 1 sesquiterpenesynthase fold with the active site in an open conformation. The metalbinding motifs were identified as D⁸⁰DQFD and N²¹⁸DVRSFAQE. Some bound water moleculeswere evident in the X-ray crystal structure, but none were obviouslypositioned to quench a putative final carbocation intermediate. Incubationsin H₂¹⁸O generated labeled product, confirmingthat the alcohol functionality arises from nucleophilic capture ofthe final carbocation by water originating from solution. Site-directedmutagenesis of amino acid residues from both within the metal bindingmotifs and without identified by sequence alignment with aristolochenesynthase from Aspergillus terreus generated mostlyfunctional germacradien-4-ol synthases. Only GdolS-N218Q generatedradically different products (∼50% germacrene A), but no directevidence of the mechanism of incorporation of water into the activesite was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F₃-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDPgenerated 12,13-(E)-β-farnesene upon beingincubated with GdolS, suggesting stepwise formation of the germacrylcation during the catalytic cycle. Incubation of GdolS with [1-²H₂]FDP and (R)-[1-²H]FDP demonstrated that following germacryl cation formation a [1,3]-hydrideshift generates the final carbocation prior to nucleophilic capture.The stereochemistry of this shift is not defined, and the deuteronin the final product was scrambled. Because no clear candidate residuefor binding of a nucleophilic water molecule in the active site andno significant perturbation of product distribution from the replacementof active site residues were observed, the final carbocation may becaptured by a water molecule from bulk solvent.

Terpenoidsmake up the largestfamily of natural products with many thousands of known compounds,spanning a wide range of biological activities, including pigments,semiochemicals, and medicinal compounds active against infectiousdiseases and many cancers.¹⁻⁶ Typically, the biosynthetic generation of mature terpenoids occursin two distinct phases. Mg²⁺-dependent cyclization of alinear isoprenyl diphosphate by a terpene synthase to generate a hydrocarbonproduct with a high degree of stereocomplexity is followed by an oxidativephase, whereby the cyclic hydrocarbon undergoes P450-dependent oxidationsand other transformations to yield highly complex terpenoids withmultiple stereocenters and a high degree of functionality.^7,8

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

According to a postulated mechanism forthe GdolS-catalyzed conversionof 1 to 2 (Figure 1), the enzyme must first guide the ionizedsubstrate through at least two carbocation intermediates before addingwater at position 4 of the cyclic carbocation intermediate 4 and at the same time prevent the addition of water to 3 and the loss of a proton from 4 and 3.Here, we have used a combination of site-directed mutagenesis, incubationof GdolS with FDP analogues, kinetic measurements, and X-ray crystallographyto investigate the mechanism of the GdolS-catalyzed conversion ofFDP (1) to (−)-germacradien-4-ol (2). We demonstrate that the cyclization mechanism proceeds via ionizationof FDP prior to cyclization, and it is proposed that the final quenchingof the carbocation cascade is conducted by a molecule of water derivedfrom bulk solvent. This water molecule could be trapped in the activesite upon substrate binding, or it could access the active site atthe end of the catalytic cycle if enabled by loop movements.

Figure 1

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

Materials and Methods

ProteinPreparation and Purification

The expressionvector containing the gene for GdolS (pET16b-SC1) was a generous giftfrom Y. Ohnishi (University of Tokyo, Tokyo, Japan). pET16b-SC1 containedthe gene for GdolS with an in-frame sequence encoding an N-terminaldecahistidine tag, permitting purification by nickel affinity chromatography.²⁰Escherichia coli BL21(DE3)cells were transformed with pET16b-SC1, and a single transformed colonywas used to inoculate 100 mL of LB medium containing ampicillin (100mg L^–1); this was grown overnight at 37 °Cwhile being shaken. This starter culture was used to inoculate LBmedium containing ampicillin (5 mL of culture per 500 mL of LB medium).The cultures were incubated at 37 °C until an OD₆₀₀ of 0.6 was reached, at which time gene expression was induced withisopropyl β-d-1-thiogalactopyranoside (IPTG, 0.2 mM)and the culture allowed to grow for a further 3 h. Cells were harvestedby centrifugation (3400g for 10 min), and the supernatantwas decanted and discarded. Pellets were thawed and resuspended in25 mL of cell lysis buffer [50 mM Tris, 100 mM NaCl, and 10 mM imidazole(pH 8.0)] and cells lysed by sonication (5 min, 40% amplitude, pulse5 s on and 10 s off). The cell debris was discarded after centrifugation(38000g for 30 min) and the supernatant applied toa Ni-NTA affinity column (QIAGEN, 5 mL). Proteins were eluted witha 10 to 500 mM imidazole gradient in lysis buffer. Fractions containingGdolS [>95% pure as judged by sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE)] were combined, dialyzed against10 mM Tris (pH 8.0), and concentrated to 10 mL. Protein concentrationswere measured using the method of Bradford^21,22 and aliquots of GdolS stored at −20 °C.

The QuikChangesite-directed mutagenesis kit (Stratagene) was used to introduce thedesired mutations following the manufacturer’s instructionsusing pairs of mutagenic primers (Table S1). Homology modeling of GdolS was conducted using the SWISS-MODELworkspace²³⁻²⁵ based on the crystal structure of a pentalenene synthasemutant [PS-N219L, Protein Data Bank (PDB) 1HM7].²⁶

Steady-state kinetic parameters were measured at 30 °C usingradiochemical assays modified slightly from those used to determinekinetic parameters in other sesquiterpene synthases.²⁷⁻²⁹ Reactions (final volume of 250 μL) were initiated by the additionof enzyme (30 nM) to assay buffer solutions [50 mM HEPES, 2.5 mM MgCl₂, and 5 mM 2-mercaptoethanol (pH 8.0)] containing 0–10μM [1-³H]FDP (24000 dpm μM^–1) and overlaid with 1 mL of hexane. After incubation for 10 min,reactions were quenched by addition of EDTA (50 μL, 0.5 M) andmixtures vortexed for 30 s. The hexane was decanted and the sampleextracted with hexane and Et₂O (11:1) in the same way (2× 750 μL). The pooled organic extracts were passed througha short column of silica (∼500 mg) into 15 mL of Ecoscint fluid(National Diagnostics), and the silica was then washed with a furtherportion of hexane and Et₂O (750 μL) and analyzedby scintillation counting. Inhibition assays were performed in anidentical fashion except for the addition of fluorinated FDPs at concentrationsranging from 0 to 10 μM.

Crystallization and StructureDetermination

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).

The cell pellet was resuspended in Ni-NTA (QIAGEN)wash buffer [50 mM K₂HPO₄, 300 mM NaCl, 10%(v/v) glycerol, and 3 mM tris(2-carboxyethyl)phosphine hydrochloride(TCEP) (pH 7.7)], and cells were lysed by sonication (10 min, 50%amplitude, pulse 1 s on and 2 s off). Cell debris was discarded aftercentrifugation (38000g for 30 min), and the supernatantsolution was loaded on a Ni-NTA column (QIAGEN, 5 mL). Protein waseluted with a 0 to 400 mM imidazole gradient in Ni-NTA wash buffer.The GdolS fractions from the Ni-NTA column were combined, concentrated,and loaded onto a 10 mL HiTrap Q HP column (GE Healthcare) after exchangewith Q wash buffer [10 mM Tris and 10 mM NaCl (pH 8.0)]. Elution wasperformed with a 0 to 500 mM NaCl gradient in Q wash buffer. Fractionscontaining GdolS (>95% pure as judged by SDS–PAGE) wereloadedonto a HiLoad Superdex 200 PG column pre-equilibrated with 137 mMsodium chloride, 2.7 mM KCl, 10 mM Na₂HPO₄,1.8 mM KH₂PO₄, and 5% (v/v) glycerol (pH 7.4).Again, fractions were analyzed by SDS–PAGE and the purest combined,concentrated to 5.5 mg/mL, and stored at −80 °C.

For crystallization, GdolS was treated with trypsin for 10 minat room temperature prior to setting up crystallization trays (1000:1GdolS:trypsin molar ratio). Crystallization was achieved by the sitting-dropvapor diffusion method at room temperature. Typically, a 1 μLdrop of protein solution [5 mg/mL GdolS, 137 mM NaCl, 2.7 mM KCl,10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, and 5% (v/v) glycerol (pH 7.4)] was added to a 1 μL dropof precipitant solution [0.1 M Tris and 2.0 M ammonium sulfate (pH8.5) (Hampton Research)] and equilibrated against an 80 μL reservoirof precipitant solution. Crystals generally formed within 1 week.

After growing to maximal dimensions, crystals of unliganded GdolSwere transferred to a cryoprotectant solution (25% glycerol and 75%mother liquor) and flash-cooled in liquid nitrogen. X-ray diffractiondata to 1.50 Å resolution were collected at beamline 4.2.2 ofthe Advanced Light Source (Lawrence Berkeley National Laboratory),indexed and integrated with Denzo in HKL2000, and scaled with SCALA.^31,32 Crystals of the unliganded protein belonged to space group P2₁: unit cell parameters a =51.4 Å, b = 74.1 Å, and c = 82.5 Å, with two monomers in the asymmetric unit and a Matthewscoefficient (V_M) of 2.1 Å³/Da (42% solvent content).

The crystal structure of unligandedGdolS was determined by molecularreplacement using Phaser for Molecular Replacement in the CCP4 package³³ using a monomer of pentalenene synthase (PDBentry 1PS1)as a search probe.³⁴ Iterative cycles ofrefinement and manual adjustment of the model were performed withPHENIX and COOT, respectively.^35,36 Water molecules wereadded to the model in the later stages of refinement. The followingdisordered segments were characterized by broken or missing electrondensity and accordingly were omitted from the final model: T158–Q161and S227–V232 (chain A) and M1–T5, G160, Q161, and A224–D231(chain B). Final refinement statistics are listed in Table 1.

Table 1

Data Collectionand Refinement Statisticsof Unliganded GdolS

resolution limits (Å)	55.1–1.50
space group	P2₁
unit cell parameters
a, b, c (Å)	51.4, 74.1, 82.5
α, β,γ (deg)	90.0, 91.7, 90.0
total no. of reflections	346240
no. of unique reflections	98277
redundancya	3.5 (3.2)
R_mergea,b	0.070 (0.978)
R_pima,c	0.044 (0.629)
CC_1/2a,d	0.998 (0.588)
I/σ(I)a	11.6 (1.2)
completeness (%)a	99.4 (99.8)
no. of reflectionsused in work set/test set	98143/1994
R_worka,e	0.157 (0.269)
R_freea,e	0.200 (0.305)
no. of protein atomsf	4977
no. of solvent atomsf	658
no.of sulfate ionsf	10
root-mean-square deviation
bonds (Å)	0.015
angles (deg)	1.3
average B factor (Å²)
main chain	21
side chain	24
solvent	38
SO₄^– anion	76
Ramachandranplot (%)
allowed	96.6
additionally allowed	3.4
generously allowed	0.0
disallowed	0.0

aNumbers in parentheses refer tothe highest-resolution shell of data.

bR_merge for replicate reflections. R = ∑|I_h – ⟨I_h⟩|/∑⟨I_h⟩, where I_h is the intensity measure for reflection h and ⟨I_h⟩ is the average intensityfor reflection h calculated from replicate data.

cR_pim = ∑[1/(n – 1)]^1/2|I_h – ⟨I_h⟩|/∑⟨I_h⟩,where n is the number of observations (redundancy).

dCC_1/2 = σ_τ²/(σ_τ² +σ_ε²), where σ_τ² is the true measurement error variance and σ_ε² is the independent measurement error variance.

eR_work = ∑||F₀| – |F_c||/∑|F₀| for reflectionscontained in the working set. R_free =∑||F₀| – |F_c||/∑|F₀| for reflectionscontained in the test set held aside during refinement (5% of total).|F₀| and |F_c| are the observed and calculated structure factor amplitudes, respectively.

fPer asymmetric unit.

Molecular Modeling

The substratemolecule (3R)-nerolidyl diphosphate (8) was geometry-optimizedin its chair conformation, followed by a single-point calculationat the Hartree–Fock level of theory with a standard 6-31G basisset, and subsequent population analysis. The LUMO molecular orbitalwas selected for the sake of illustrating the mechanism of allylicbond reshuffling as it displays the expected alternation of orbitalcoefficients after ring closure. The diphosphate group was replacedby OH for the sake of convenience of calculation. The calculationswere performed using GAUSSIAN09.³⁷

Results

Structureof GdolS

Unliganded GdolS-E248A adopts theα-helical fold characteristic of a class I terpenoid synthase,as first observed in the crystal structure of avian farnesyl diphosphatesynthase.³⁸ GdolS crystallizes as an isologousdimer in the asymmetric unit through the interactions of helices E,F, H, and I. As often observed for dimeric terpenoid cyclases,³⁹ the active sites of the GdolS dimer are orientedin antiparallel fashion. The H-1α loop, the loop following theNSE motif, and the F–G loop are not resolved because of thehigh mobility of these regions (Figure 2).

Figure 2

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.

Sequence alignments with other terpene synthasesshow that twometal binding motifs D⁸⁰DQFD and N²¹⁸DVRSFAQE are conserved (boldface indicates presumptive metal bindingligands). The crystal structure of GdolS shows that these segmentsflank the mouth of the active site on helices D and H, respectively.While the penultimate residue in the second metal binding motif isusually an arginine or a lysine residue, GdolS has a glutamine atthis position. The function of this residue is to hydrogen bond withthe substrate diphosphate group, so it is interesting to note thesubstitution of the shorter, uncharged side chain for the longer,positively charged side chain that is normally conserved for thisfunction in other sesquiterpene synthases.⁴⁰ An omit electron density map showing the metal binding motifs inunliganded GdolS is shown in Figure 3.

Figure 3

Simulated annealing omit map calculated for the metalbinding segmentsD⁸⁰DQFD and N²¹⁸DVRSFAQEin GdolS (wall-eyed stereoview contoured at 2.5σ).

The size of the GdolS active site, approximately15 Å deepand 10 Å wide, is comparable to that of other sesquiterpene synthases.The unliganded enzyme crystallized in the open conformation as hadalso been observed in the crystal structure of unliganded aristolochenesynthase from Penicilium roqueforti (PR-AS)⁴¹ and several monomers of aristolochene synthasefrom Aspergillus terreus (AT-AS).¹⁰ The active site is predominantly nonpolar with a contourdefined by aliphatic and aromatic side chains. Curiously, althoughGdolS utilizes a water molecule to quench the final carbocation intermediatein the cyclization cascade, the active site does not contain any polarside chains that could readily serve as a general base to facilitatethe reactivity of a trapped water molecule. In the structure of theopen active site conformation presented here, a number of orderedsolvent molecules are observed in the GdolS active site. However,we cannot conclude that any of these water molecules play specificstructural or catalytic roles.

Metal Binding Residues

To probe individual amino acidresidue contributions to the GdolS-catalyzed conversion of FDP to(−)-germacradien-4-ol (2), various residues werereplaced using site-directed mutagenesis and product distributionswere analyzed by gas chromatography and mass spectrometry (GC–MS)of organic extractable products. Steady-state kinetics were measuredfor all functional mutants.

Single-amino acid replacements inthe aspartate-rich region, specifically D80, D81, and D84, with glutamatecreated functional GdolS variants with only minor effects on the productdistribution (Table 2). Each of these enzymes produced minor amounts of germacrene A (5) and D (6) (total <2%) in the pentane extractableproducts, and while functional, the measurement of the steady-statekinetic parameters showed that these GdolS variants were significantlyless active than the wild-type enzyme. GdolS-D80E was too slow todetermine steady-state kinetic values. The GdolS-D81E- and GdolS-D84E-catalyzedreactions had turnover numbers, k_cat,approximately 10-fold lower than that of the wild-type enzyme. Eachmutation had little effect on the binding of FDP to the enzymes (K_M values of 0.82 ± 0.11 and 0.92 ±0.09 μM, respectively).

Table 2

Steady-State KineticParameters andProduct Distributions (%) for the Conversion of FDP

			productdistribution
enzyme	k_cat (s^–1)	K_M (μM)	germacradien-4-ol (2)	germacreneA (5)	germacrene D (6)
GdolS-WT	0.079 ± 0.003	1.07 ± 0.13	100	–	–
GdolS-D80E	ndaa	ndaa	>98	<1	<1
GdolS-D80N	n/ab	n/ab	n/ab	n/ab	n/ab
GdolS-D81E	0.010 ± 0.0006	0.82 ± 0.11	>99	<1	<1
GdolS-D81N	ndaa	ndaa	>99	<1	–
GdolS-D84E	0.009 ± 0.0003	0.92 ± 0.09	>98	<1	<1
GdolS-D84N	n/ab	n/ab	n/ab	n/ab	n/ab
GdolS-E226D	ndaa	ndaa	>99	<1	–
GdolS-S222A	ndaa	ndaa	>98	–	<2
GdolS-N218Q	ndaa	ndaa	>47	>50	<2
GdolS-N218L	ndaa	ndaa	>99	<1	–
GdolS-N218T	n/ab	n/ab	n/ab	n/ab	n/ab
GdolS-N218E	n/ab	n/ab	n/ab	n/ab	n/ab

aNo detectableactivity; activitytoo low for kinetic parameters to be determined.

bNot applicable.

Replacement of D80, D81, and D84 with asparagine ledto inactiveenzymes with only GdolS-D81N showing small amounts of activity. Productscould be observed in the pentane extracts, but activity was too lowto allow the measurement of kinetic parameters. These results areconsistent with previously reported effects of amino acid replacementsin the metal binding domains, with replacement of the first aspartatein the DDXXD motif (D80 in GdolS) having a much greater effect oncatalytic activity than replacement of the others.^26,42,43 This is also consistent with molecular modelingresults reported by van der Kamp et al.,¹¹ who predicted that upon formation of the Michaelis complex two ofthe requisite Mg²⁺ ions are coordinated to this first aspartateof AT-AS.

Replacement of N218 of the NSE motif of GdolS withglutamine generateda functional enzyme that converted FDP to almost identical quantitiesof germacradien-4-ol (2) (47.6%) and germacrene A (5) (50.7%), in addition to a small amount of germacrene D(6) (1.7%). However, this substitution had a detrimentalimpact on the catalytic efficiency and the steady-state kinetic parameterscould not be measured. Because the amino acids of the NSE motif chelateone of the three Mg²⁺ ions required for catalysis, it islikely that the longer ligand side chain perturbs ideal metal coordinationgeometry and thus perturbs the catalytic function of the Mg²⁺₃ cluster. In an attempt to further shift the productdistribution toward germacrene A (5), N218 was also replacedwith leucine, a residue with similar shape and volume but with nometal coordination or hydrogen bonding capability. With the loss ofa critical metal coordination interaction in the NSE motif, as wellas the loss of possible hydrogen bond interactions with the N218 sidechain, coupled with a corresponding increase in hydrophobicity throughthe N218L substitution, it was proposed that this might prevent ingressof water into the active site and further reduce the proportion ofgermacradien-4-ol produced. GdolS-N218L was an inefficient enzymethat produced >98% germacradien-4-ol and only traces of germacreneA (5), so water still had ready access to the activesite in this mutant. However, the catalytic activity of GdolS-N218Lis severely compromised. Other replacements of N218 with amino acidsof varying degrees of hydrogen bond donors and acceptors (threonineand glutamate) yielded inactive proteins.

Origin of the HydroxylGroup of 2 and PotentialWater-Directing Residues

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.

Y303 was replaced sequentially with F, I, and T.GdolS-Y303F was generated to test whether the phenolic OH was responsiblefor hydrogen bonding with the nucleophilic water molecule, while GdolS-Y303Iremoves hydrogen bonding capability altogether. The side chain ofT is an alternative hydrogen bond donor/acceptor equivalent. However,both F and I replacements of Y303 yielded functional enzymes withonly minor effects on product distribution while GdolS-Y303T was inactive.Indeed, it appears that Y303 has a greater role in the stabilizationof carbocation intermediates given the dramatic reduction of catalyticactivity observed for GdolS-Y303I.

Replacement of E307 withQ produced an enzyme that was an effectiveGdolS, and only traces of germacrene A (5) and germacreneD (6) side products were observed. GdolS-E307Q was approximately10 times less efficient than the wild-type enzyme (Table 3). Replacement of the same residuewith methionine had little effect on product distribution, albeitwith a dramatic loss of catalytic efficiency.

Table 3

Steady-StateKinetic Parameters andProduct Distribution (%) of Wild-Type GdolS and Y303 Mutants

			productdistribution
enzyme	k_cat (s^–1)	K_M (μM)	germacradien-4-ol (2)	germacreneA (5)	germacrene D (6)
GdolS-WT	0.079 ± 0.003	1.07 ± 0.13	100	–	–
GdolS-Y303I	ndaa	ndaa	>99	trace	–
GdolS-Y303F	0.014 ± 0.006	0.66 ± 0.12	>96	>2	<1
GdolS-Y303T	n/ab	n/ab	n/ab	n/ab	n/ab
GdolS-E307Q	0.013 ± 0.0004	2.24 ± 0.16	>98	<1	<1
GdolS-E307M	ndaa	ndaa	>95	>4	<1

aNo detectable activity;activitytoo low for kinetic parameters to be determined.

bNot applicable.

Figure 4

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-PP_i,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.

Incubations of GdolS withFDP Analogues

To investigatethe catalytic mechanism of GdolS with regard to the diphosphate ionizationand subsequent carbocationic reaction cascade, the wild-type enzymewas incubated with various FDP analogues. The nature of the initialcarbocation was explored with the fluorinated FDP analogues 2-F-FDP(1b), 15,15,15-F₃-FDP (1c), and12,13-F₂-FDP (1d), which might intercept putativering closure steps through the destabilization of carbocation intermediates.Incubation of 1b and 1c with GdolS generatedno detectable products in the pentane extracts as judged by GC–MSanalysis, even under extended incubation times. The effect of theseFDP analogues as inhibitors was investigated by measuring steady-statekinetic parameters in the presence of varying amounts of fluorinatedFDP. Both 2-F-FDP and 15,15,15-F₃-FDP were found to bepotent inhibitors of GdolS with measured K_i values of 1.1 ± 0.56 and 3.9 ± 0.2 μM, respectively.Double-reciprocal plots indicated that the mode of inhibition waslargely noncompetitive, suggesting that they may only partially occupythe active site upon binding. The binding interactions of prenyl diphosphateswith a terpene synthase are most likely dominated by the interactionof the negatively charged diphosphate group with the positively chargedmagnesium ions, and it is not surprising that the K_i values are comparable to substrate K_M values. These results may suggest that the initial formationof germacryl cation from FDP proceeds in a stepwise fashion throughlinear farnesyl cation prior to 1,10-ring closure (Figure 5) because the electron-withdrawingfluorine atoms on C2 and C15 increase the energy of the allylic farnesylcation and thereby prevent its formation. In previous work, we observedthat the terpene cyclases PR-AS⁴⁴ and δ-cadinenesynthase from Gossypium arboreum(45) are capable of converting 2F-FDP to cyclic products, indicatingthat an S_N2-like synchronous cyclization can take placefor this substrate; this result strongly suggests that farnesyl cationformation is required for GdolS.

To further probe this, GdolSwas incubated with 12,13-F₂-FDP (1d), a substrateanalogue that should not prevent farnesyl cation formation becausethe fluorine atoms are located on the distal isopropylidene groupbut will prevent 1,10-ring closure because the π-electron densityon C10 and C11 is reduced, thereby lowering the double bond nucleophilicityand increasing the energy of a putative cation with the charge onC11.⁴⁶ Incubation of 1d withGdolS generated a single pentane extractable product, 12,13-difluoro-(E)-β-farnesene (7), as judged by GC–MS. 7 was identified by comparison with the product generatedupon incubation of 1d with (E)-β-farnesenesynthase. This result lends strong support to the proposal of a stepwiseformation of germacryl cation because this must arise from loss ofdiphosphate from the abortive substrate and subsequent loss of a protonfrom C15 to generate the farnesene product.

Incubation of GdolSwith nerolidyl diphosphate (NDP, 8), often proposed asa reactive intermediate in sesquiterpene synthase-catalyzedreactions,^45,47 produced germacradien-4-ol asa single product. Measurement of the steady-state kinetic parameterswith racemic [³H]NDP⁴⁸ gavevalues for k_cat and K_M of 0.076 ± 0.003 s^–1 and 1.3± 0.03 μM, respectively, remarkably similar to those measuredfor FDP (Table 4).Use of enantiopure (3R)-[³H]NDP⁴⁹ for the measurement of kineticparameters showed a 3-fold increase in k_cat paired with a 2-fold increase in K_M (0.24± 0.007 s^–1 and 2.6 ± 0.18 μM, respectively),leading to a modest improvement in catalytic efficiency over thatof the substrate FDP (1).

Table 4

Comparisonof Steady-State KineticParameters for the GdolS-Catalyzed Turnover of FDP, Racemic [³H]NDP, and (3R)-[³H]NDP

1-³H-labeledsubstrate	k_cat (s^–1)	K_M (μM)	k_cat/K_M (s^–1 μM^–1)
FDP	0.079 ± 0.003	1.07 ± 0.13	73.8 × 10^–3
racemic[³H]NDP	0.076 ± 0.003	1.30 ± 0.03	58.5 × 10^–3
(3R)-[³H]NDP	0.240 ± 0.007	2.60 ± 0.18	92.3 × 10^–3

To probe thenature and stereochemistry of the hydride shift followinggermacrenyl cation formation, GdolS was incubated with [1-²H_n]FDP analogues. GdolS was incubatedwith [1-²H₂]FDP [1e (Figure 5)], and the pentane extractableproducts were analyzed by GC–MS. The mass spectrum of the resulting[²H₂]germacradien-4-ol (Figure S23) contained a molecular ion at m/z 224 consistent with the incorporation of bothdeuterons. The dehydrated molecular ion fragment [M – H₂O]⁺ showed an ion at m/z 206, indicating that it is also doubly deuterated. Thefragment derived from the loss of the isopropyl group shows an ionat m/z 162, indicating that a singledeuterium atom has been lost and only one remains on the remainingring fragment, hence supporting a [1,3]-hydride shift mechanism (Figures 1 and 5); consecutive [1,2]-hydride shifts, as observed for (R)-germacrene D synthase,⁵⁰ wouldleave both deuterons on this fragment (m/z 163). The stereochemistry of the 1,3-hydride shift wasalso investigated through the incubation of GdolS with (R)-[1-²H]FDP (1f). Interestingly, analysisof the mass spectrum of germacradien-4-ol arising from this showedfragments at both m/z 161 and 162,with no clear enrichment in either, e.g., as is evident in the sameexperiment with δ-cadinene synthase.⁴⁵ This indicates that the hydride shift is not stereospecific possiblydue to a small degree of rotation around the C10, C11 bond that wouldallow a shift of either hydride. Alternatively, there may be scramblingof the stereochemical information through equilibration of the stabilizedallylic carbocation and the germacryl cation, which would lead tono overall isotopic enrichment of either species upon fragmentationof the isopropyl group.

Discussion

GdolS, first discoveredthrough genome mining of Streptomycescitricolor,²⁰ provides an excitingcase study for examining how sesquiterpene synthases generate alcoholproducts through an exquisitely selective carbocationic reaction cascadeduring which the high-energy and reactive intermediates must be shelteredfrom the aqueous medium yet subsequently generate the product throughnucleophilic capture of the final carbocation with a molecule of water.

Attempts to form cocrystals of GdolS with diphosphate, Mg²⁺, and farnesyl diphosphate analogues were frustratingly unproductivein this study, but diffracting crystals of unliganded apo-GdolS wereobtained. The 1.50 Å resolution structure revealed that GdolSfolds as a classic class 1 sesquiterpene synthase with the metal bindingmotifs identified as D⁸⁰DQFD and N²¹⁸DVRSFAQE.Some ordered water molecules were observed in the active site of thisstructure, but with the active site in an open conformation and disorderpreventing observation of the complete H-1α and F–G loops,it is not clear whether these waters play a role in catalysis. Thereare no obvious active site residues that could hydrogen bond withor activate a nucleophilic water molecule. Site-directed mutagenesisof the metal binding residues in the DDXXD and NSE motifs resultedin either inactive enzymes or functional mutants with little changein product distribution. Only GdolS-N218Q showed a significant perturbationof the product distribution with 51% germacrene A (5)formed alongside 48% germacradien-4-ol (2) and some germacreneD (6). Changes in the Mg²⁺_B coordinationpolyhedron presumably caused by substitution of the larger glutamineresidue for the asparagine ligand to Mg²⁺_B inwild-type GdolS most likely reorient the diphosphate group in theactive site so that instead of playing a spectator role, it may prematurelyquench the germacryl cation by acting as general base for removalof a proton from C12 or C13 to generate germacrene A (5) (Figure 5). Incubationof wild-type GdolS with FDP in the presence of labeled water confirmedthe incorporation of a solvent water molecule (Figure S8). There is a precedent for active site-trapped watermolecules in bornyl diphosphate and aristolochene synthases,^14,15 and this was believed to be the most likely source of the incorporationof water by GdolS. However, we have found no evidence of any residuesinvolved in the direct incorporation of an active site water molecule.An alternative hypothesis is that the final carbocation is simplyquenched by a molecule of water from the bulk solvent. With the carbocationstill bound in the active site, specific movements of the H-1αand E–F loops restrict water molecules to approach from the re face of the final carbocation, resulting in the stereo-and regiospecific formation of (−)-germacradien-4-ol (2).

Incubation of GdolS with the fluorinated FDP analogues2-F-FDP(1b) and 15,15,15-F₃-FDP (1c)yielded no detectable products, and both compounds proved to be potentnoncompetitive inhibitors. Incubation of 12,13-F₂-FDP (1d) with GdolS produced exclusively 12,13-difluoro-(E)-β-farnesene (5), suggesting a stepwiseformation of germacryl cation via an intermediate farnesyl cation(Figure 5). Incubationof GdolS with deuterated analogues [1-²H₂]FDP(1e) and (R)-[1-²H]FDP (1f) demonstrated that the [1,3]-hydride shift in the nextstep of the catalytic cycle occurs with scrambling of the stereochemistryat C1.

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.

Taken together, the results reportedhere suggest that after theformation of the Michaelis complex the GdolS-catalyzed conversionof FDP (1) to (−)-germacradiene-4-ol (2) proceeds through farnesyl cation and rapid 1,10-ring closure togermacrenyl cation. The subsequent rapid 1,3-hydride shift yieldsa stabilized allylic carbocation, which is then quenched by a watermolecule just before product release. The mechanism of water captureis unclear, but in the absence of evidence of an active involvementof residues from GdolS, enzyme loop movements may be required to facilitateingress of water to quench the final carbocation.

Figure 5

Reaction outcomes catalyzedby GdolS in this study. The wild-typereaction is colored red. Abortive, potential products, not generated,are colored gray. (a) Reaction catalyzed by wild-type GdolS from FDP(1a). (b) Alternative (minor) pathway observed for someGdolS mutants resulting in germacrene D. (c) Alternative (minor) pathwayobserved for some GdolS mutants resulting in germacrene A. Panel Ashows products arising from incubation of GdolS with deuterated FDPanalogues.

Figure 6

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

Acknowledgments

We thankProf. Yasuo Ohnishi (University of Tokyo) for plasmidpET16b-SC1 harboring the cDNA of GdolS, Dr. Juan A. Faraldos for helpfuldiscussions, and Dr. Rob Jenkins, Robin Hicks, Simon Waller, and ThomasWilliams (Cardiff University) for assistance with mass spectrometryand NMR. We thank Jay Nix for help with data collection at beamline4.2.2 at the Advanced Light Source. The Advanced Light Source is supportedby the Director, Office of Science, Office of Basic Energy Sciences,of the U.S. Department of Energy under Contract DE-AC02-05CH11231.

Supporting Information Available

The Supporting Informationis available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00115.

Synthesis of FDPanalogues, protein characterization,table of mutagenic primers, gas chromatograms from enzymatic incubations,mass spectra, and kinetic data (PDF)

Supplementary Material

bi6b00115_si_001.pdfbi6b00115_si_001.pdf

Accession Codes

The atomiccoordinatesand crystallographic structure factors of GdolS-E248A have been depositedin the Protein Data Bank as entry 5I1U.

This work wassupported by the U.K.’s Biotechnology and Biological SciencesResearch Council (BBSRC) through Grants BB/H01683X/1 and BB/G003572/1, the U.K.’s Biotechnology and BiologicalSciences Research Council (BBSRC), the U.K.’s Engineering andPhysical Sciences Research Council through Grant EP/L027240/1, theCardiff Synthetic Biology Initiative, Cardiff University, and NationalInstitutes of Health Grant GM56838.

The authorsdeclare no competing financial interest.

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