Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis

Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in k(cat) that was offset by a 50-fold decrease in K(m), resulting in a relatively minor 2-fold decrease in catalytic efficiency, k(cat)/K(m). The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).     

Mechanism and stereochemistry of the germacradienol/germacrene D synthase of Streptomyces coelicolor A3(2)

Incubation of farnesyl diphosphate (1, FPP) with recombinant germacradienol synthase from Streptomyces coelicolor A3(2) gave, in addition to (4S,7R)-germacra-1(10)E,5E-diene-11-ol (2), 15% of (-)-germacrene D (5). Incubations of [1,1-2H2]FPP (1a), (1R)-[1-2H]FPP (1b), and (1S)-[1-2H]FPP (1c) with germacradienol/germacrene D synthase and analysis of the resulting samples of germacradienol (2) and germacrene D (5) by a combination of 1H, 2H, and 13C NMR and mass spectrometry established that it is H-1si of FPP that is lost in the formation of germacradienol (2) and that undergoes 1,3-hydride transfer in the formation of (-)-germacrene D (5). The proportion of the two products was also sensitive to isotopic labeling, with cyclization of (1S)-[1-2H]FPP (1c) giving an increased proportion (35%) of 5. These results could be explained by a mechanism involving partitioning of a common helminthogermacradienyl cation intermediate 7.     

Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.     

Effect of isotopically sensitive branching on product distribution for pentalenene synthase: support for a mechanism predicted by quantum chemistry

Mechanistic proposals for the carbocation cascade reaction leading to the tricyclic sesquiterpene pentalenene are assessed in light of the results of isotopically sensitive branching experiments with the H309A mutant of pentalenene synthase. These experimental results support a mechanism for pentalenene formation involving a 7-protoilludyl cation whose intermediacy was first predicted using quantum-chemical calculations.     

Templating effects in aristolochene synthase catalysis: elimination versus cyclisation

Analysis of the products generated by mutants of aristolochene synthase from P. roqueforti (PR-AS) revealed the prominent structural role played by the aliphatic residue Leu 108 in maintaining the productive conformation of farnesyl diphosphate to ensure C1-C10 (σ-bond) ring-closure and hence (+)-aristolochene production.     

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

Beta-D-Xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D: -xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), and 1,4-beta-D-xylobiose (X2) was determined on and off (k (non)) the enzyme at pH 5.3, which lies in the pH-independent region for k (cat) and k (non). Rate enhancements (k (cat)/k (non)) for 4NPX, 4NPA, and X2 are 4.3 x 10(11), 2.4 x 10(9), and 3.7 x 10(12), respectively, at 25 degrees C and increase with decreasing temperature. Relative parameters k (cat) (4NPX)/k (cat) (4NPA), k (cat) (4NPX)/k (cat) (X2), and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(X2) increase and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(4NPA), (1/K (m))(4NPX)/(1/K (m))(4NPA), and (1/K (m))(4NPX)/(1/K (m))(X2) decrease with increasing temperature.     

Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin

Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.     

Cuprate-Mediated Synthesis and Biological Evaluation of Cyclopropyl- and tert-Butylfarnesyl Diphosphate Analogs

The novel farnesyl diphosphate (FPP) analog 3-cyclopropyl-3-desmethylfarnesyl diphosphate (3-cpFPP, 1) was designed as a potential mechanism-based inhibitor of the FPP-utilizing enzyme protein-farnesyl transferase (PFTase). The key step in the synthesis of 1 involved the stereoselective coupling of vinyl triflate 8 with a lower order cyclopropyl cyanocuprate to afford the desired cyclopropyl ester 13. The sterically encumbered analog 3-desmethyl-3-tert-butylfarnesyl diphosphate (3-tbFPP, 7) was synthesized via a similar route. The use of the more reactive higher order tert-butyl cyanocuprate led to lower yields of ester 11, the key intermediate in the synthesis of 7. Biological evaluation of 3-cpFPP demonstrates that it is not a time-dependent inhibitor of recombinant yeast PFTase. Instead, 3-cpFPP is an alternative substrate for this enzyme that exhibits a K(m) comparable to FPP and a k(cat) only 5-fold lower than the natural substrate. In contrast, 3-tbFPP is an exceptionally poor substrate for yeast PFTase and acts as an inhibitor of this enzyme.     

Inhibition of (+)-aristolochene synthase with iminium salts resembling eudesmane cation

Trigonal iminium halides of (4aS,7S)-1,4a-dimethyl- and (4aS,7S)-4a-methyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium ions, aimed to mimic transition states associated with the aristolochene synthase-catalyzed cyclization of (-)-germacrene A to eudesmane cation, were evaluated under standard kinetic steady-state conditions. In the presence of inorganic diphosphate, these analogues were shown to competitively inhibit the enzyme, suggesting a stabilizing role for the diphosphate leaving group in this apparently endothermic transformation.     

Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

Proton inventory studies of alpha-thrombin-catalyzed reactions of substrates with selected P and P' sites

Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P(1)-P(3) sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.1 and (DOD)(k(cat)/K(m)) = (0.8-2.1) +/- 0.1 and (2) internally fluorescence-quenched substrates (a) (AB)Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys(DNP)-Asp-OH, an optimal sequence, and (b) (AB)Val-Ser-Pro-Arg-Ser-Phe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII, are (DOD)k(cat) = 2.2 +/- 0.2 and (DOD)(k(cat)/K(m)) = (0.8-0.9) +/- 0.1, at the pL (L = H, D) maximum, 8.4-9.0, and (25.0-26.0) +/- 0.1 degrees C. The most plausible models fitting the partial isotope effect (proton inventory) data have been selected on the basis of lowest values of the reduced chi squared and consistency of fractionation factors at all substrate concentrations, assuming rate-determining acylation. The data for Z-Pro-Arg-7-AMC are consistent with a single-proton bridge at the transition state phi(TS) = 0.39 +/- 0.05 and components for solvent reorganization phi(S) = 0.8 +/- 0.1 and phi(S) = 1.22 for k(cat) and k(cat)/K(m), respectively. The data for tripeptide amides fit bowl-shaped curves; an example is N-t-Boc-Val-Pro-Arg-7-AMC: phi(TS)(1) = phi(TS)(2) = 0.57 +/- 0.01 and phi(S) = 1 for k(cat) and 1.6 +/- 0.1 for k(cat)/K(m). Proton inventories for the nonapeptide (2b) are linear. The data for k(cat) for H-D-Phe-L-Pip-Arg-pNA and the decapeptide (2a) are most consistent with two identical fractionation factors for catalytic proton bridging, phi(TS)(1) = phi(TS)(2) = 0.68 +/- 0.02 and a large inverse component (phi(S) = 3.1 +/- 0.5) for the latter, indicative of substantial solvent reorganization upon leaving group departure. Proton inventory curves for k(cat)/K(m) for nearly all substrates are dome-shaped with an inverse isotope effect component (phi(S) = 1.2-2.4) originating from solvent reorganization during association of thrombin with substrate. These large contributions from medium effects are in full accord with the conformational adjustments required for the fulfillment of the dual, hemostatic and thrombolytic, functions of thrombin.     

Quantitative analysis of the effect of salt concentration on enzymatic catalysis.

Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt-rate profiles: k(cat)/K(M) = (k(cat)/K(M))(MAX)/[1+([Na+]/K[Na+])(n')], where (k(cat)/K(M))(MAX) is the physical limit of k(cat)/K(M), K(Na+) is the salt concentration at which k(cat)/K(M) = (k(cat)/K(M))(MAX)/2, and -n' is the slope of the linear region in a plot of log(k(cat)/K(M)) versus log [Na+]. The value of n' is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with k(cat)/K(M) values that can exceed 10(9) M(-1) s(-1). Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n'. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n'. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n', as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt-rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis.     

Bifunctional abietadiene synthase: free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.     

Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study

In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)βk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.     

Genome mining in Streptomyces clavuligerus: expression and biochemical characterization of two new cryptic sesquiterpene synthases

Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His?-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-2H?]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1(si) of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1(si) of FPP.     

Fundamental reaction mechanism for cocaine hydrolysis in human butyrylcholinesterase

Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.     

Transient kinetic studies of protein hydrolyses by endo- and exo-proteases on a 27 MHz quartz-crystal microbalance

We have compared endo- and exo-type protease reactions and characterized the enzymatic reaction mechanisms by determining all kinetic parameters (k(on), k(off), k(cat), K(d) = k(off)/k(on), and K(m) = (k(off) + k(cat))/k(on)) by following the mass change of the formation and the decay of the enzyme-substrate (ES) complex (k(on) and k(off)), and the formation of the product (k(cat)) on a 27 MHz quartz-crystal microbalance in aqueous solutions. The K(m) value was nearly equal to the K(d) value for the endo-type protease (subtilisin and alpha-chymotrypsin); however, in the case of exo-type protease (carboxypeptidase P), the K(m) value was quite different from the K(d) value, due to k(cat) > k(off).     

Inhibitor scaffolds as new allele specific kinase substrates

The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N(6)-(benzyl)-ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was preferred by v-Src (T338G) (k(cat)/K(M) = 3.2 x 10(6) min(-)(1) M(-)(1)) over ATP or the previously described ATP analogue, N(6) (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (k(cat)/K(M) = 2.6 x 10(4) min(-)(1) M(-)(1)) comparable to ATP (k(cat)/K(M) = 5.0 x 10(4) min(-)(1) M(-)(1)) largely due to a significantly better K(M) (6.4 microM vs 530 microM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N(6)-(benzyl)-ATP with respect to the wild-type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma-(32)P]-3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, even while competing with cellular levels (4 mM) of unlabeled ATP. The fact that this new more highly orthogonal nucleotide is accepted by three widely divergent kinases studied here suggests that it is likely to be generalizable across the entire kinase superfamily.     

Stereochemistry of eudesmane cation formation during catalysis by aristolochene synthase from Penicillium roqueforti

The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital and density functional theory calculations, a proton transfer mechanism has recently been proposed, in which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2) proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the elimination and protonation reactions was investigated by the analysis of the reaction products from incubation of 1 and of [12,12,12,13,13,13-(2)H(6)]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H(2)O and D(2)O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of with PR-AS in D(2)O led to the production of (6R)-[6-(2)H] aristolochene, indicating that protonation occurs from the face of the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds, along which protons could be delivered from the solvent to the active site.     

Structures and Biosynthesis of Corvol Ethers—Sesquiterpenes from the Actinomycete Kitasatospora setae

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one‐ and two‐dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.