Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti

The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.     

Structural basis for antibody catalysis of a cationic cyclization reaction

Antibody 4C6 efficiently catalyzes a cationic cyclization reaction. Crystal structures of the antibody 4C6 Fab in complex with benzoic acid and in complex with its eliciting hapten were determined to 1.30A and 2.45A resolution, respectively. These crystal structures, together with computational analysis, have elucidated a possible mechanism for the monocyclization reaction. The hapten complex revealed a combining site pocket with high shape complementarity to the hapten. This active site cleft is dominated by aromatic residues that shield the highly reactive carbocation intermediates from solvent and stabilize the carbocation intermediates through cation-pi interactions. Modeling of an acyclic olefinic sulfonate ester substrate and the transition state (TS) structures shows that the chair-like transition state is favored, and trapping by water directly produces trans-2-(dimethylphenylsilyl)-cyclohexanol, whereas the less favored boat-like transition state leads to cyclohexene. The only significant change observed upon hapten binding is a side-chain rotation of Trp(L89), which reorients to form the base of the combining site. Intriguingly, a benzoic acid molecule was sequestered in the combining site of the unliganded antibody. The 4C6 active site was compared to that observed in a previously reported tandem cyclization antibody 19A4 hapten complex. These cationic cyclization antibodies exhibit convergent structural features with terpenoid cyclases that appear to be important for catalysis.     

Unearthing the roots of the terpenome

Although terpenoid synthases catalyze the most complex reactions in biology, these enzymes appear to play little role in the chemistry of catalysis other than to trigger the ionization and chaperone the conformation of flexible isoprenoid substrates and carbocation intermediates through multistep reaction cascades. Fidelity and promiscuity in this chemistry (whether a terpenoid synthase generates one or several products), depends on the permissiveness of the active site template in chaperoning each step of an isoprenoid coupling or cyclization reaction. Structure-guided mutagenesis studies of terpenoid synthases such as farnesyl diphosphate synthase, 5-epi-aristolochene synthase, and gamma-humulene synthase suggest that the vast diversity of terpenoid natural products is rooted in the facile evolution of alpha-helical folds shared by terpenoid synthases in all forms of life.     

Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate

The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in complex with substrate analogues geranyl-S-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 and 1.95 ? resolution, respectively. This terpenoid cyclase catalyzes the cyclization of the naturally occurring, noncanonical C-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C(11) homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of (+)-bornyl diphosphate synthase and endo-fenchol synthase, plant cyclases that convert geranyl diphosphate into products with closely related bicyclic bornyl skeletons, but distinct structures and stereochemistries.     

Molecular recognition of the substrate diphosphate group governs product diversity in trichodiene synthase mutants

The X-ray crystal structures of Y305F trichodiene synthase and its complex with coproduct inorganic pyrophosphate (PP(i)) and of Y305F and D100E trichodiene synthases in ternary complexes with PP(i) and aza analogues of the bisabolyl carbocation intermediate are reported. The Y305F substitution in the basic D(302)RRYR motif does not cause large changes in the overall structure in comparison with the wild-type enzyme in either the uncomplexed enzyme or its complex with PP(i). However, the loss of the Y305F-PP(i) hydrogen bond appears to be compensated by a very slight shift in the position of the side chain of R304. The putative bisabolyl carbocation mimic, R-azabisabolene, binds in a conformation and orientation that does not appear to mimic that of the actual carbocation intermediate, suggesting that the avid inhibition by R- and S-azabisabolenes arises more from favorable electrostatic interactions with PP(i) rather than any special resemblance to a reaction intermediate. Greater enclosed active-site volumes result from the Y305F and D100E mutations that appear to confer greater variability in ligand-binding conformations and orientations, which results in the formation of aberrant cyclization products. Because the binding conformations and orientations of R-azabisabolene to Y305F and D100E trichodiene synthases do not correspond to binding conformations required for product formation and because the binding conformations and orientations of diverse substrate and carbocation analogues to other cyclases such as 5-epi-aristolochene synthase and bornyl diphosphate synthase generally do not correspond to catalytically productive complexes, we conclude that the formation of transient carbocation intermediates in terpene cyclization reactions is generally under kinetic rather than thermodynamic control.     

Exploring biosynthetic diversity with trichodiene synthase

Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP(i)). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.85A resolution X-ray crystal structure of the complex with Mg(2+) (3)-PP(i) and the benzyl triethylammonium cation, which is a bulkier mimic of the bisabolyl carbocation intermediate in catalysis. Taken together, these findings show that the active site of trichodiene synthase is sufficiently flexible to accommodate bulkier and electronically-diverse substrates and intermediates, which could indicate additional potential for the biosynthetic utility of this terpenoid cyclase.     

Mechanism-Based Inhibitors and Other Active-Site Targeted Inhibitors of Oxidosqualene Cyclase and Squalene Cyclase

Abstract

Enzymatic cyclizations of squalene and oxidosqualene lead to sterols and other triterpenoids in bacteria, fungi, plants, and animals. The cyclases for these reactions catalyze formation and stabilization of polycyclic carbocations and direct the enzyme-specific, templated formation of new carbon-carbon bonds in regio- and stereochemically defined contexts. The development of mechanism-based irreversible inhibitors, photoactivatable inhibitors, and numerous substrate analogs have helped to unravel the stepwise events occurring in the catalytic sites of these enzymes by covalent modification of specific amino acid residues.     

细菌中倍半萜生物合成的研究进展

萜类化合物是一大类小分子天然产物,在生物体内扮演重要的角色。植物和真菌中萜类化合物的生物合成已被广泛研究,但是在真核生物中克隆或改造萜类化合物生物合成途径还有较大难度。许多细菌同样可以产生萜类化合物。在过去十多年间细菌萜类合酶的研究进展为我们对萜类化合物生物合成的理解做出了显著的贡献。这里我们主要关注细菌中合成的倍半萜化合物,概述其化学结构、倍半萜合酶对法尼基焦磷酸环化的机制、后修饰酶特别是氧化还原酶所参与的后修饰、代谢调控以及合成途径中尚未解决的问题等。     

Point mutation of (+)-germacrene A synthase from <Emphasis Type="Italic">Ixeris dentata</Emphasis>

Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine. To probe the carbocation-stabilizing role, the amino acid residues of the ₄₃₃TSA₄₃₅ triad in (+)-germacrene A synthase from Ixeris dentata were altered by site-directed mutagenesis. Enzyme kinetic measurements of the mutants and GC/MS analysis of the enzyme reaction products indicate that mutations of the triad decreased enzyme catalysis rather than substrate binding but did not affect its structural rearrangement in the catalytic mechanism. This is the first report that the hydroxyl group of threonine at the first position of the triad is required for the cyclase activity.     

X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.     

Carbocations in the synthesis of prostaglandins by the cyclooxygenase of pgh synthase? a radical departure!

Evidence already available is used to demonstrate that although prostaglandin G/H synthase hydroxylates arachidonic acid through radical intermediates, it effects cyclizations through a carbocation center at C-10. This is produced following migration of H to the initial radical at C-13 and a 1epsilon oxidation. Under orbital symmetry control, the cyclizations can give only the ring size and trans stereochemistry actually observed. After cyclization, the H-shift reverses to take the sequence back into current radical theory for hydroxylation at C-15. Thus 10,10-difluoroarachidonic acid cannot be cyclized, although it can be hydroxylated. Acetylation of Ser516 in the isoform synthase-2 is considered to oppose carbocation formation and/or H-migration and so prevent cyclizations while permitting hydroxylations; the associated inversion of chirality at C-15 can then readily be accommodated without the change in conformation required by other schemes. Suicide inhibition occurs when carbocations form stable bonds upon (thermal) contact with adjacent heteroatoms, etc. Because the cyclooxygenase and peroxidase functions operate simultaneously through the same heme, phenol acts as reducing cosubstrate for the cyclooxygenase, thus enabling it to promote PGG2 production and protect the enzyme from oxidative destruction.     

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.     

Conserved tyr residues determine functions of Alicyclobacillus acidocaldarius squalene-hopene cyclase

The catalytic cavity of Alicyclobacillus acidocaldarius squalene-hopene cyclase is mainly lined by aromatic amino acids. In recombinant cyclases, three out of four tyrosine residues (Y) have been mutated to phenylalanine residues (F). The mutant cyclases Y495F and Y612F had less activity than the wild-type cyclase, but a wild-type product pattern. Mutant Y609F had wild-type activity but a drastically altered product pattern with hopene and significant amounts of bicyclic alpha-polypodatetraene and different tetracyclic triterpenes (dammaradienes and eupha-7,24-diene). The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.     

Alternative termination chemistries utilized by monoterpene cyclases: chimeric analysis of bornyl diphosphate, 1,8-cineole,and sabinene synthases

Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile. Exchange of smaller sequence elements identified a region of roughly 70 residues from 1,8-cineole synthase that, when substituted into sabinene synthase, conferred the ability to produce 1,8-cineole. A similar strategy identified a small region of bornyl diphosphate synthase important in conducting the anti-Markovnikov addition to the bornane skeleton. Observations made with these chimeric monoterpene cyclases are discussed in the context of the recently determined crystal structure for bornyl diphosphate synthase.     

Characterization of the adenylation site in the RNA 3'-terminal phosphate cyclase from Escherichia coli

RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human and Escherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA, yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3'-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation.     

Structure of geranyl diphosphate C-methyltransferase from Streptomyces coelicolor and implications for the mechanism of isoprenoid modification

Geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a noncanonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl S-thiolodiphosphate (GSPP) along with S-adenosyl-L-homocysteine in the cofactor binding site, resulting from in situ demethylation of S-adenosyl-L-methionine, at 2.05 or 1.82 ? resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. S-Adenosyl-L-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor S-adenosyl-L-methionine. These structures provide important clues about the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation-π interactions.     

Activation-independent cyclization of monoterpenoids

The biosynthesis of cyclic monoterpenes (C(10)) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C(30)), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase from Zymomonas mobilis (ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.     

Structural diversity of the triterpenic hydrocarbons from the bacterium Zymomonas mobilis: the signature of defective squalene cyclization by the squalene/hopene cyclase

Twelve polycyclic triterpenic hydrocarbons (alpha- and gamma-polypodatetraenes, dammara-20(21),24-diene, 17-isodammara-12,24-diene, eupha-7,24-diene, hop-17(21)-ene, neohop-13(18)-ene, 17-isodammara-20(21),24-diene, neohop-12-ene, fern-8-ene, diploptene and hop-21-ene) were detected in the hydrocarbon fraction from the bacterium Zymomonas mobilis. Some of them have never been reported from bacteria. These triterpenes were present in Z. mobilis in significant amounts, comparable to those of diploptene, which is usually the major triterpenic hydrocarbon in hopanoid-producing bacteria. The occurrence of such compounds confirms the lack of specificity of bacterial squalene cyclases and the possibility of alternative cyclization routes induced by the existence in the cyclization process of intermediate carbocations of sufficient lifetime.     

Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita).

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.     

Chemical synthesis of terpenoids with participation of cyclizations plus rearrangements of carbocations: a current overview

Many terpenoids are biosynthesized after a cascade of cyclizations and rearrangements of carbocations mediated by terpenoid synthases, as exemplified in th