Structural Biology and Chemistry Cf the Terpenoid Cyclases

3412 C/II~III,nav. LVVB~ I VO, 0- i L-J-L~L

Structural Biology and Chemistry cf the Terpenoid Cyclases

David W. Christianson*

Roy and Diana Vagelos Laboratories, Depafiment of Chemistry, University of Pennsyivania 231 South 34th Street Philadelphia, Pennsylvania 19104-6323

Received Gciobsr 2;

Contents I. 'Introduction 2, Monoierpene Cyclases 3. Sesquiterpene Cyclases 3.1. Trichodiene Synthase 3.2. Epi-Aristolochene Synthase 3.3. Aristolochene Synlhase 3.4. Pentalenene Synthase 4. Biterpene Cyclases 4.1. Abietadiene Synthase 4.2. Taxadiene Synthase 5. Priterpene Cyclases 5.1. Squalene-Hopene Cyclase 5.2. ~xidos~ualene~yclke 3432 Born in Attieboro, Massachusetts, in 1961, David W. Christians017rec 6. Antibody Catalysis of Cationic Cyclization 3433 the A.B. (1983), A.M. (1985), and Ph.D. (1987) degrees in chemis, Reactions Harvard, where he studied in the research group of William N, hip 6.1. Catalytic Antibody 19A4 3434 Jr. Christianson joined the faculty of the University of Pennsylv 1988, where he is currently the Roy and Diana Vagelos Profe 6.2. Catalytic Antibody 466 3437 Chemistry and Chemical Biology. Christianson's research focuses 7. Concluding Remarks 3433 structtlres and mechanisms of metal-requiring enzymes. 8. Acknowledgments 344'1 9. References 3441 and carbocation intermediates throueh- a rerles of n:-ec1.s conformations leading to one unique cycllzatlon p:oducl in contrast. other terpenord cyclases are rather p,*'o~il:substrate uct formation could reflect inco~noleteevolutioil of a isreclre undergo changes in bonding, I~ybridization,or configuration active site template in these cyclases.

during the course of an intricate cyclization cascade initiated The terpene cyclase substrates geranyl d~phosphaie(Clo) by the formation and propagation of highly reactive car- famesyl diphosphate (CI5),geranylgeranyl dlphosphale (Cro: bocation intermediates.'-'0 Tens of thousands of terpene and geranylfmesyl diphosphate (G25)(171gu1-e 1) each derrxre natural products have been chavacterized to date, yet each from a chain elo~lgatiolireaction catalyzed by an lsov ultimately derives froni one of only a handful of linear diphosphate synthase (also known as a prenyltrajzsfe isoprenoid substrates (Figure I). Terpene biosynthesis thus utilizing the five-carbon precursors dimethylallyl diphcsp 1 provides an elegant example of Nature's strategy for com- and isopentenyl diphosphate.I2 Tlie 30-carbon substi' binatorial chetnical synthesis and diversity. squalene derives from the head-to-head condensat~oriof ln ' Most terpenoid cyclases generate a unique hydrocarbon molecules of farnesyl dlphosphate m a reactlor? catalyzei product with remarkable structural and stereochemical preci- by squalene syntliase.I3 '"emarkably. the eiuy;i?e\ iild' sion. For exalnpIe. consider the reaction cataIyzed by generate terpene cyclase substrates are themselves st~~clii~a!l' oxidosqualene cyclase, also known as lanosterol synthase, related to class I teipene cyclases (Figure 2), ~ndrcarlve'-i in the biosynthesis of cholesterol (Figure 1). Only 1 out of their divergence from a common primordial aizcesior edll\ a possible 128 lanosterol stereoisomers is formed in the in the evolution of tespene biosynthes~s.This it~iiciurJ~ ii cyclization of the 30-carbon triterpene (8-2,3-oxidosqualene, homology is seminiscer~tof slrggestions that enzj lie: ii'l so the enzyme active site clearly serves as a high-fidelity catalyze successive steps in biosynthetic pathway ma) zllalc template that chaperones the flexible polyisoprenoid substrate common evolutionary origins.I5 r Given its prominence as the archetypical prenyltra~~sf~j~~c*:, * Phone: 215-898-5714. Fax: 215-573-2201. E-maii: [email protected]. and given the pro~i~nentstructural and funct~ona!ho17?oh?j~~ ii 10.1 021icr050286w CCC: $59.00 O 2006 American Chemical Soc~ety Pubilshed on Web 06/01/2006

@#$%D1511020215HG00*&^% Linear Terpenes Examples of Cyclic Terpenes

dimethyialiyi diphosphate isopentenyl diphosphate

Drmethyallyl ~PP- -OPP - J-opv 1 -pp, I ' geranyl diphosphate A AOPP "OH A qP? + Lopp -PP, (-) menthol (i)borny diphosphaie --

geranyifarnesyl diphosphate

mangicol G scalarin

lanosterol hopene gnre 1. Cene~alscheme of terpeno~dnomenclature and blosynthesls (OPP = diphosphate, PP, = inorganic diphosphate)

tween the prenyltransferases and the terpenoid cyclases, second aspastate-rich motif on helix H, Asp-244, coordinates phate synthase provides a suitable introduction to Mg2+l. for understanding tespenoid cyclase structure The structure of the ternary enzyme-substrate-substrate 1 diphosphate synthase is a analogue con1plex2' provides a foundation for u~lderstanding subunits with a core of 10 the electrophilic alkylation mechanism proposed for iso- unding a hydrophobic active site of ap- prenoid chain elongation in which the metal-triggered 213 (Figures 3 and 4).16 Helices D ionization of dimethylallyl diphosphate yields an allylic o-called "aspai-tate-rich" motifs, DDXXD, carbocation-pyrophosphate ion followed by alky- alytic function.I7-l9 The structure of the lation of the C3-C4 x bond of isopentellyl diphosphate to pe enzymeI6 and the structure of the Phe- yield a tertiary C3 carb~cation;~~27 stereospecific elimination -Ala/Phe-1 13-Ser variant complexed with geranyl of a C2 prot0n,2~,~~proposed to be mediated by a pyropkios- the first residue in the first aspartate- phate oxygeaZ2terminates the chain elongation reaction to , makes a syrz,syn-bidentate coordination yield geranyl diph~sphate.~~A second condellsation reaction Mg2+ ions, and one carboxylate oxygen with the addition of isopentenyl diphosphate to geranyl last residue in this motif, Asp-121, makes a s)m,aizti- diphosphate yields product farnesyl diphosphate (Figure 7). n interaction between the two metal ions Interestingly, famesyl diphosphate synthase can be engi- neered to generate Ionger chain products such as geranyl- recent crystal structure deter~~nationof farnesyl geranyl diphosphate by site-directed ~nutagenesisof aromatic yhate synthase from Escizeviclzia coli complexed with residues at the base of the active site cleft; the deeper active entenyl diphosphate and the nonreactive sub- site cleft allows for the synthesis of a Ionger prod~ct.~~,~~ ethylallyl-S-thiolodiphosphate2' reveals Like the prenyltransferase reaction, the terpenoid cyclase lete trinuclear magnesium cluster neacrion is initiated by the formation of a highly reactlve ai-tate-rich motifs and the diphosphate carbocation. The terpenoid cyclases utilize two main chem- thylallyl-S-thiolodiphusphate[Figure 6).2"he cal strategies for initial carbocation formation: (Ij metal- 2+2 ions each form six-membered ring chelate triggered departure of a pyrophosphate (PP,) leaving group. s with thiolodiphosphate oxygen atoms. The first similar to the strategy employed by farnesyl diphosphate of the first aspartate-rich motif, Asp-105 on helix synthase, or (2) protonation of an epoxide ring or a carbon- es syn,sy;lz-bidentate coordination interactions with carbon double bond, a strategy employed by oxidosqualene and Mg2+3. One car-boxylate oxygen of the third cyclase or squalene-hopene cyclase, respectively. Thus, the tate in the motif, Asp-Ill, bridges Mg2'2 and Mg2+3 initiation step of a terpene cyclase reaction is either ioniza- " anti-coordination stereochemistry. Notably, the first tion-dependent or protonation-dependent. -rich motif in the bacterial enzyme is of the form Ionization-dependent terpenoid cyclases-monotespene, XD. this contrasts with the avian enzyme, in which sesquiterpene, and ditespene cyclases-contain an aspastate- istic DDXXD pattern is observed. Regardless, rich sequence, DDXXDIE, on helix D that is topologically last aspmates of the first aspartate-rich motif identical to the first such sequence occurring in the prenyl- e make identical interactions with a pair of transferases on helix D, but the second such sequence on res 5 and 6). The first aspartate in the helix H is not "aspartate-rich" in the terpenoid cyclase: it

@#$%D1511020215HG00*&^% Figure 3. Dimer of avian farnesyl diphosphate synthase. a- are represented by cylinders. Helices of monomer 1 ! with capital letters and connected by orange loops. and ilel monorner 2 are labeled with primed capital letters and col by magenta loops. The 2-fold axis of the dirner is approx vei-tical, and the two active site clefts are oriented in parallel and open at the "top" of the nlolecule. Reprinted with pen from ref 16. Copyright 1994 Arnerican Cherilical Societj-. F

Squalene Synthase Richodiene Synthasa

Figure 2. Structural similarities among various terpenoid synthases (red) define the core class I terpenoid cyclase fold. The product of each synthase is indicated. At the bottom, rms deviations of Ca atoms and the number of structmally similar residues (in paren- Figure 4. Ribbon plot of the avian fasnesyl dipliosphate Sj'il theses) are listed. Reprinted with permission from ref 60. Copyright Two aspartate-rich motifs. Asp- I 17-Asp-!21 and Asp-257 2001 National Academy of Sciences, U.S.A. 261 (red), flank the mouth of the active site.'"

has diverged to a consensus sequence of (L,V)(V,L,A)- domains, and each catalyzes a discrete (N,D)D(L,I,V)X(S,T)XwxE (boldface residues are magne- cyclization of geranylgeranyl diphosphate t sium ligands, so this motif is also designated the "NSEDTE" (section 4.1). motif).31 Both motifs together bind a trinuclear magnesium It should be noted that the initiation step of cluster that triggers the departure of the substrate diphosphate synthase can result in direct carbocation forn leaving group, as fusther outlined in sections 2, 3.1, and 3.2. In contrast, protonation-dependent terpenoid cyclases- certain diterpene cyclases and triterpene cyclases-contain subsequently reacts with the G2-C3 x-bor a single signature sequence DXDD, in which the central diphosphate (Figure 7). Alternatively, the il aspastate residue is implicated as the proton donor that be accompanied by direct attack of one of the I- triggers initial carbocation formation.1° bonds at the site of incipient cai-bocatio~iformaiio The common a-helical fold of the ionization-dependent with leaving group departure to form a carbocatic terpenoid cyclases (Figure 2) is designated the class I distant carbon center (SKPreaction"). terpenoid cyclase fold, and the unrelated a-bari-el fold of may open in either Markovnikov fashion (formi the protonation-dependent cyclases (discussed in section 5) stable carbocation) or anti-Markovnikov fashioi is designated the class II tespenoid cyclase fold.6 Interest- the less stable carbocation); Not ingly, plant cyclases such as (+)-bomyl diphosphate synthase roninellt of the active site can contr (section 2) or epi-aristolochene synthase (section 3.2) contain both domains, but only the class I domain is the catalytically active terpenoid cyclase. Strikingly, the ditespene cyclase abietadiene synthase from grand fir also contains both the cyclization of an isoprenoid diphosph

@#$%D1511020215HG00*&^% mes for initial carbon-carbon transiently forned carbocation intermediates through dipole- 1 diphosphate ionization charge interactions, certain fixed and protected dipoles in equent reaction of the an enzyme active site can similarly stabilize carbocation bonds with the four differellt carbocation irtteimediates. Additionally, carbocation reaction intermedi- in Figure 8: followed by hydride shifts, methyl ates can be stabilized by the ring n electrons of aron~atic potentially vast and diverse residues such as phenylalanine, tyrosine, and tryptophan n of farnesyl diphosphate. through cation-n or charge-quadrupole interactions (Fig- tes thus firmly implicated in ures 9 and

ilieu of an enzyme active for ionization-dependent monoterpene and sesquiterpene ues or being quenched by cyclases in sections 2 and 3, Although no diterpene cyclase short, the hydrophobic prenyltransferase or has yet yielded a crystal structure, important structure- ase active site serves as an "inert organic solvent" mechanism analogies can be made for this family of terpene tion-mediated chain elongation or cycljzation cyclases based on the structures of monotelrpene and ses- ation reactions are facili- quiterpene cyclases, and these are reviewed in section 4. It , aprotic organic solvents capable of stabilizing is especially intriguing that the bifunctional diterpene cyclase

@#$%D1511020215HG00*&^% Fign~~9, (a) The side chain of an aroma --&ib'ribFd as an electroiiic quadrupole, the char are no net charge and opposed dipoles. E potentials for benzene, phenol, and indole (b. c) of these aromatic rings bear significant positive Figure 7. Chain elongation reaction catalyzed by famesyl diphos- potential..and the same is expected for the core phate synthase: ionization of geranyl diphosphate (a) leads to the of the amino acids phenylalanine, tyrosi formation of the corresponding allylic cation. which then undergoes respectively (red = negative; blue = positive condensatioll with isopentenyl diphosphate (b) to yield a tertiary in a terpene synthase active site may stabilize reac C3 carbocation, from which stereospecific elimination of a proton intermediates without the danger of reacting (c) yields farnesyl diphosphate. PP = diphosphate; PP, = inorganic ates. Reprinted with permission from Scierzce (http:l11 pyrophosphate. Reprinted with permission from ref 25. Copyright ref 34. Copyright 1996 American Association for the Ad: snc 1981 American Chemical Society. of science.

AE = -7.9 hcaiin~ul OPLS-AA AE = -1 1 5 bi.il/iinoi Figure 8. Four possible initial ring closure reactions for farnesyl AE = -8 0 hciii/niol 6-31e AE = -12 0 kalinu! diphosphate (FPP). the substrate of a sesquiterpene cyclase. By enforcing a particular conformation of the flexible isoprenoid Figure 10. Optimized geometries calculated with the OP substrate. the enzyme active site plays a critical role in directing force field for catioil-zr interactions between the 2-metiiyi-3 initial carbon-carbon bond formation to generate tertiary carbocation and benzene (left) and indole (right). Stabilizatioi? e cation intermediates (Markovnikov addition to a double bond, for complex formation from the separated cation and iniii- pathways a and c) or less stable secondary carbocation intermediates thermodynamically favorable and are calculated usirig rilrez (anti-Markovnikov addition to a double bond, pathways b and d). computational procedures. The optimal geometries of these Pathways a and b require initial ionization and isomerization to cal cation-z interactions can easily be achieved in a form nerolidyl diphosphate, reionization of which allows for the cyclase active site for cation interactions with the ;r systems incorporation of a cis double bond in the six-membered or seven- side chains of phenylalanine, tyrosine, or tryptophan. Reprint membered ring, respectively. Reprinted with permission from ref permission from ref 35. Copyright 1997 American Cli 33. Copyright 1995 American Chemical Society. Society.

abietadiene synthase contains separate ionization-dependent monotenpenes and their derivatives are largely respa and protonation-dependent active sites. The tritelrpene cy- for the pleasing fragrances and flavors of aromatic p clases are exclusively protonation-dependent, and squalene- (+)-Bornyl diphosphate synthase, a inonoterpeize c~ hopene cyclase and oxidosqualene cyclase are reviewed in from Salvia officinalis (culinary sage), is a I28 kD I section 5. In section 6, progress in the generation of novel dimer that catalyzes an unusual cyclization cascade ' antibody catalysts of isoprenoidpolyene cyclization reactions the PP;leaving group of geranyl diphosphate is rein is discussed, and I conclude in section 7 with comments on rated into the final cyclization product (Figure emerging stsucture-mechanism relationships common to the Positional isotope exchange studies with 180-labeled sa greater family of terpenoid cyclases. demonstrate that the same phosphoester oxyger a contained in the prenyl diphosphate linkages of su 2. Monoterpevse Cyc!ases geranyl diphosphate and product (+)-bornyl diphosplxat suggesting very precise positioning and orientatioii Each member of the family of monoterpene cyclases diphosphate moiety throughout the cyclization cascad catalyzes the metal-dependent ionization and cyclization of is covalently bound or ion-paired with substrate, inle the linear 10-carbon isoprenoid precursor geranyl diphosphate ates, and product. Mechanistic feetures of the cycli (Figure 1). Although monoterpene cyclases catalyze the cascade have been carefully delineated in enzyme: simplest terpene cyclization cascades in that they utilize the studies (Figure 1 2) ."7-4' shortest linear isoprenoid cyclization substrate, these enzymes The X-ray crystal structure of (+)-bornyl dl nevestheless achieve remarkable structural and stereochem- synthase4' reveals a C-terminal domain that ical diversity in their product arrays (Figure 11)."he volatile characteristic class I tenpene cyclase fold: this

@#$%D1511020215HG00*&^% (+)-bornyl dipkosphale

1 diphosphate by monoterpene cyclases yields a diverse array of n~onocyclicand bicyclic products nyl carbocation intermediate. Reprinted with kind permission of Springer Science and Business Media from ref yright 2000 Springer-Verlag.

-dependent cyclization of geranyl diphosphate is cata- aspartate-rich motif, Asp-35 1, bridges alld ~~pwith (Figure 13). The N-terminal giycosyl hydr~lase-like syrz,syn-bidentate coordination stereochemistry, whereas one d function, although the N-terminal alorn of the third asf;artate, A~~-35%,bridges M~F eptide caps the active site in the C-terminal domain and ~g;' with syiz,anti-coordination stereochemistry. The group of the subsuate NSEDTE appevis as Asp-496, Thr-500, and Glu-S()J, rogeranyl diphosphate, the PPi in ternary complex with 7-aza-7,X-dihydrol'tmonene and these residues chelate ~g;+on the opposite side of the zabomane, or product (+)-bornyl diphosphate (Figure active site 'left. One Oxygen of the fi-phosphate of Ihe inal polypeptide segment is involved in substrate analogue and product bridges ~gi*and IVIg;'; n bond interactions with the C-terminal and a second oxygen coordinates to ~g;+.One oxygen two with the aspartate-rich motif (kg- atom of the a-phosphate coordinates to ~g:? and a secolld Asp352). Interestingly, oxygen coordinates to ~g?such that two six-membered rg-56 of (+)-bOrll~l di~hos~hate ring cllelate interactions are formed between &e diph0sphae-e scent of tandem arginines found in~the group, ~gr,+,and ~g?(Figure 14). la1 segments of several (but not all) plant tel-pene Truncation studies with lirnonene synthase impli- Intriguingly, the constellation of diphosphate-metal in- es in the isomefization teractions observed in (+)-bornyl diphosphate synthase is it is conceivable that the tandem identical to that observed in diphosphate complexes with E.

-56 of (+)-bonlyl &phosphate synthase. A it is tempting to speculate that this .diphosphate molecular is considered for diter-ene cyclases contain- recognilion scheme corresponds to the universal molecular r Lys-Arg pair in the N-terminus recognition of prenyl diphosphates by class I terpencid cyctases. However, the binding of the trinuclear magnesium (+)-bornyl diphasphate synthase duster lo the sesquiterpene cyclase trichodiene synthase is phates or PPi and aza andogues slightly divergent, in that only one residue in the aspartate- iates reveal essentially identical rich segment pa~icipatesin metal binding (section 3.1); metal the diphosphate moiety involving binding to epi-aristolochene synthase exhibits additional agnesium cluster and several hydrogen bond differences (section 3.2). Possibly, the divergence of metal (Figure 14).~?The first aspartate residue in the binding and prenyl diphosphate recognition by the binuclear

@#$%D1511020215HG00*&^% For any cyclization reaction involving geranjil diPl it is clear that diphosphate ionization must be fojio isoinerization to a cisoid linalyl intermediate to i$. cyclization to form the a-terpinyl cation (the trans double bond of geranyt diphosphate would otherwi geranyl diphasphale 3-sza-2,3-dihydra- / (+)-barmy[ diphasphale (GFP) geranyl diphasphate I [BPP) formation of the six-membered ring) (Figure 12). I I 4 metal-activated diphosphate departure, isomerirati~~to linalyl diphosphate allows rotation about the C2-C.3 to the cisoid conformer, which re-ioizizes to . cyclizako-n by 61-66 ring cIosure to form th - -.. terpiny! cation-PPi ion pair (Figure 16). Cation tions with Phe-578 and Trp-323 appear to stabilize carbocation. Subsequent anti-Markovniltov cyciizatjor the secondary 2-bornyl cation, which is lleuiralir stereospecific C-O bond formation with PPi on the face to yield (+)-bornyl diphosphate. The binding orientations and conformations of sxl- analogue 3-aza-2,3-dihydrogeranyldiphosphate ai- a-tespinyl cation analogue 7-aza-7.8-dihydrolimone properly mimic the productive conformations re catalysis, possibly due in part to overwhelming electrostatic and hydrogen bond interactions involvi positively charged aza nitrogen atoms. However. the b conformation of 2-azabornane in the ternary complex PP, nearly matches that of the product (+)-bornyl di Figure 12. The cyclization of geranyl diphosphate to form (+)- phate. Taken together, these results seem to imply th bornyl diphosphate proceeds through intermediate (3R)-linalyl more product-like the carbocation analogue is. the diphosphate and the (4R))a-terpinyl and 2-bornyl carbocation likely it is to be bound in a catalytically prod intermediates. The diphosphate ester oxygen of the substrate (red) is retained in the diphosphate ester of the product. Aza analogues conforniation. This hypothesis is also consisle~lt\vi of carbocation inteimediates are illustrated in boxes. Reprinted with ailomalous binding modes of aza-analogues cornpiexed permission from ref 43. Copyright 2002 National Academy of trichodiene synthase, discussed in section 3.1. Sciences. U.S.A.

magnesium cluster reflects an additional strategy in the Sesquitespene cyclases catalyze the metal-depeade evolution of diverse terpenoid cyclases. clizatioii of farnesyl dipl~osphatein the generation of Comparisons of experimentally determined structures of than 300 known monocyclic, bicyclic, and tricyclic complexes between (+)-bornyl diphosphate synthase and aza u~ts.',~.~.~Bacterial and fungal enzymes such as perltal 1

analogues of carbocation intermediates (compounds 1-3 in synthase from Str-eptonzyces UG5319 and aristn!~ I Figure 12), as well as product, indicate that the hydrophobic synthase from Perzicilliur7z r-oqueforti are usually ac active site cleft serves as a template to chaperone reactive -40 kD single-domain monomers, but there ar I substsate and intermediate conformations through an intricate examples of homodimesic enzymes such as tricho 4 cyclization cascade while the diphosphate group is rigidly synthase from Fusar-iunz spo~*otr-iclzioides.As for C d bound.43 A variety of aliphatic and aromatic residues lining monoterpene cyclases, plant sesquiterpene cyclases ti the hydrophobic active site cavity, including Trp-323, Ile- epi-aristolochene synthase from Nicotiarzn tahaczri: J 344, Val-452, and Phe-578, ensure the proper binding an additional N-terminal domain exhibiting a fo :I conformation of the substrate and carbocation intermediates. closely related to that of the glycosyl hydrolases and w % Notably, a single water molecule, water no. 110, remains l~omologousto the class I1 tespenoid cyclase fold.' klth trapped in the active site cavity in all enzyme-analogue and there is no specific catalytic chemistry established !I8 enzyme-product complexes (Figure 15). Water no. 110 N-terminal domain of a plant sesquitei-pene cycl LC makes hydrogen bond interactions with diphosphate or the nevertheless plays a role in capping the active site gl PP, anion, the backbone carbonyl of Ser-451, and the side C-terminal class I cyclase domain. There is general1 fr ai chain of Tyr-426, so this water molecule appears to be firmly low amino acid sequence identity among seso id anchored in the active site. sufficiently so that it cannot easily cyclases from bacteria, fungi, and plants, but these cl react with carbocation intermediates to prematurely quench adopt the characteristic class I terpenoid cyclase ri. the cyclization cascade. Possibly, water no. 110 could serve contain the signature aspartate-rich and NSEIDTE rn te. as a diphosphate-assisted general base to account for the implicated in binding the b-inuclear magnesium c S t? generation of cyclic olefin side products such as pinenes, required for catalysis. In the remainder of this se tri camphene, and lim~nene.~~That the reaction of noncyclizable structure-mechanism relationships in four sesquit is( analogues of geranyl diphosphate generates significant cyclases are reviewed that provide interesting and iii;.ip re! amounts of acyclic terpenol products may reflect the presence co~nparisons with the monoterpene cyclase ( ' -bo of a trapped water molecule in the enzyme active site.45It is diphosphate synthase discussed in the pre 1 notable that a trapped water molecule can contribute to the ir contour of the active site template of a tespenoid cyclase, 3.1. Trichodiene Synthase ph and it is also notable that such a water molecule could play Trichodiene synthase is a sesquitei-pene the an essential role in catalysis. catalyzes the first committed step in the bi w"10

@#$%D1511020215HG00*&^% diphosphate synthase. Panel a shows the unliganded monomer, oriented such that the viewer is looking directly ts are labeled using the convention established for farnesyl diphosphate

mational changes that stabilize previously disordered polypeptide segnlents G-termillus of helix H and the K-a1 loop (Thr-500-Asp-50% and part of the

different trichothecene mycotoxins such as T-2 reaction. (3R)-Nerolidyl diphosphate is conclusively dem- vomitoxin, which are frequent contaminants of onstrated to be a catalytic intermediate in enzymological 6,47 The enzyme purified studies with isotopically labelled farnesyl diphosphate and oides is a dimes of 45 kD subunits nerolidyl dipho~phate.~~.~~Additionally, experiments with the nd the enzyme has been modified substrates (7s)-trans-6,7-dihydrofarnesyldiphos- rexpressedS1in Escheri- phate and (7R)-tipans-6,7-dihydrofarnesyldiphosphate yieid ase is the best-studied sesqui- product assays consistent with the formation of the corre- mechanistic enzymology and sponding 6,7-dihydronerolidyl diphosphate intermediate; chanism initially proposed for which is unable to cyclize further due to the absence of the studies with the first enzyme C6-C7 double bond.js Since farnesyl dkhosphate ionization the apple mold fungus, Trichot]zeciumroseurn, is the rate-limiting chemical step of catalysis,56alternative product assays derived from modified substsates provide a esyl diphosphate powerful demonstration of the existence of this otherwise llowed by recapture of the undetectable inter~nediate.~~ form (3R)-nerolidyl diphos- Rotation about the C2-63 bond of (3R)-nerolidyl diphos- circumvent phate 2 from a transoid to a cisoid conformation facilitates ouble bond, that SN'cyclization by C1-66 closure with PPi departure to form equent 61-C6 ring closure the conesponding bisabolyl carbocation 4 (Figure 17). The

@#$%D1511020215HG00*&^% Figure 14, Molecular recognition of diphosphate or the PP, anion by (+)-bornyl diphosphate synthase is governed by numerous coordinatiob and hydrogen bond interactions. The diphosphate position and orientation is nearly invariant in enzyme co~nplexeswith (yellow), 3-aza-2,3-dihydrogeranyldiphosphate (not shown), 7-aza-7,s-dihydrolimonene(not shown), 2-azabornane-PPi (magenta). , - bornyl diphosphate (blue). This rigid molecular recognition is consistent with the observation that the same oxygen atom conrp:.i prenyl diphosphate ester linkages of both substrate and produ~t.'~.~~Reprinted with permission from ref 43. Copyright 2002 National Ac of Sciences, U.S.A.

Figure 15. Simulated annealing omit electron density map of (+)-bornyl diphosphate synthase complexed with product (+)-bomyl dipha pc (contoured at 40). Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. No PL water molecule no. 110 remains trapped in the active site cavity along with the cyclization product. Reprinted with permission fro111r th,

Figure 16. Model of the (4R)-a-terpinyl cation hound to the active site of (+)-bornyl d~phosphatesynthase based on the bln 2-azabomane-PP, and (+)-horny1 d~phosphate The tertiary carbocation may be stabilized by cation-z interactrons w~th Phe-578 Repr~ntedwith pennlsslon from ref 43 Copyright 2002 Nat~onalAcademy of Sc~ences,U S A

cationic analogue of bisabolyl carbocation 4, (4R)-7-azabis- and Arg-304 -- Lys variants of trichodiene synthas abolepe (Figure 18), exhibits strong synergistic inhibition plexed with PPi and azabisabolene inhibitors reveai 11 with PPi (Ki = 0.51 3~ 0.07 ,LAM),but since (4s)-7-azabis- or disordered azabisabolene binding conformations, s abolene exhibits comparable behavior (Ki = 0.47 i 0.07 which appear to be driven by favorable electrostatic in pM),it is possible that stereochemical discrimitlation is u7eak tions between the negatively charged PP, anion a at the corresponding step of cataly~is.~'In fact, the X-ray positively charged alkylammonium moiety of the az

crystal structures of the Asp-100 -+ Glu, Tyr-305 - Phe, lene inhibitor (Figure 19).j8,j9Aromatic residues in tl

@#$%D1511020215HG00*&^% reactions is under kinetic rather than ther~nodynamiccon-

Further cyclization of the bisabolyl carbocation followed by a 1,4-hydride shift, tandem 1,2-methyl migrations, and final deprolonation colnpletes the biosynthesis of t~ichodiene

due is less to nificant

generation of certain aben-ant products by D lOOE tricbodiene synthase.(j1 The X-ray crystal structure of wild-type irichodielie synthase reveals that each subunit of the dimer adopts the class I terpenoid cyclase fold (Figure The quaternary structure is similar to that of the monoterpene cyclase (f)- bornyl diphosphate synthase (Figure 13) in that the two active (S@-7-azabisabolene (4s)-7-azabisabolene sites of the trichodiene synthase dimer are oriented in @R)-7-Azabisab01m is a carionic analogue of the antiparallel fashion. However. there are differeilce.; ~iith isabolyl carbocation in the trichodiene synthase mecha- e~?iintiomer,(4s)-7-azabisabolene, binds with essentiallj~ regard to the specific helices that form the dimerization 'sity in synergy with PPi bindi~g.5~ interface. It is interesting that the dimelization modes of these two cyclases further contrast with that of farnesyl diphosphate synthase, in which active sites are oriented in parallel fashion

structural changes

The binding of the trinucleas magnesium cluster in the

@#$%D1511020215HG00*&^% Figure 20. Panel a shows a stereoplot of the trichod~enesynthase monomer The aspartate-lrch motif beginning with Asp-I00 01,heii. 2 is magenta, and the NSEDTE rnotlf beginning wrth Asn-225 on helrx H IS orange A conserved baslc inotrf begrnning with Aig-3g?. ,, ,,;, J-K loop IS ~'ellow Hel~cesare labeled according to the conventron estabhshed for farnesyl dlphosphate synthase l6 Panel S

Figure 21. Panel a shows the simulated annealing omit map of the trichodiene syl~thase-Mg'+~-PP~complex contoured at 50. atomic coordinates for PPi, Mg2+ ions (gray spheres), and metal-coordinated water molecules (red spheres) are superimposed shows the hydrogen bond and nietal coordination interacsons in the trichodiene s~nthase-Mg~+~-PP~complex viewed fro111 th the active site. In panel c, superposition of unIiganded trichodiene synthase (cyan) and the trichodiene synthase-Mg2'?-P. (yellow) reveals structural changes triggered in indicated segments that cap the active site cleft. The PPi anion is magenta. 111 view looking directly into the active sites of unliganded trichodiene synthase (cyan) and the trichodiene synthase-Mg2';-P' (yellow) reveals that Asp-101 (the second residue in the conserved aspartate-rich mot$ and Arg-304 (the second arginine in the co1 basic motif) form a double hydrogen bonded salt link that stabilizes the Iigand-bound conformation. Because Arg-304 hydrogen bond to PPi, this interaction reveals how the diphosphate moiety of the farnesyl diphosphate substrate likely higgers a c change to sequester the active site from bulk solvent. Reprinted with permission from ref 60. Copyright 2001 uational Acade U.S.A. complex with tsichodiene synthase, the carboxylate group contrast with these terpene synthases, no othe of Asp-100 (the first residue in the aspartate-rich motif) the aspartate-rich segment of trichodiene syntha coordinates to and with syi~,~~n-bidentate to a metal ion. The NSEIDTE motif of trichodi geometry. The correspotlding asparlate also coordinates to (Asn-225, Ser-229, and 61u-233) chelates t n/3$ and ~g?in farnesyl diphosphate synthase (Figures (Figure 21) in a fashion similar to that obsei~edin ii- 5 and 6),20.22(+)-bornyl diphosphate synthase (Figure 14),43 diphosphate synthase (Figure 14) and epi-aris:~ and epi-aristolochene synthase (section 3.2). However, in synthase (section 3.2).

@#$%D1511020215HG00*&^% , __,. - - "EY

1- 1- i WT

i / iarnesyl diphosphate ~arnesene p-bisabolene 1

1 a-iuprenene isochernigiene a-bisabolene

2. Ami~zoacid substit~~tionsfor residues that coordinate to catal~iticmetal ions (Asp-100 - GIu) or residues that hydrogen bond

(lyr-305 -+ Phe) result in substantially broader product arrays reflecting incomplete cyclization or cyclization to alternative bicyciic "-" Reprinted with permission from ref 58. Copyright 2005 American Chemical Society.

Structure of Asp-100 -- Glu trichodiene synthase unliganded (cyan) and complexed wit11 PPi-Mg2+2 (yellow). PPi is red. The .ed active site closure is significantly attenuated in comparison with that observed for the wild-type enzyme in Figure 21~.Reprinted isson from ref 61. Copyright 2002 American Chemical Society.

en[ metal binding interactions among the terpenoid is substituted for Mg2', so it is clear that the metal Implicate subtle differences in Mg2+3-substrate coordination polyhedra play a crucial role in stabilizing the I-e interactions as a possible strategy for the proper active site contour required for productive cata~ysis.~" of product diversity. Consider. for example, site- Amino acid substitutions for residues that hydrogen bond :.iants of trichodiene synthase in which amino acid with ppi, such as Tyr-305 (Figure 21b), similarly yield ns are made in the aspartate-rich motif. The Asp- diverse product arrays. The Tyr-305 - Phe substitution has lu and Asp-101 -- Clu variants exhibit modest little effect on kcat and a minor effect on Kh4,yet this variant in KM and kc,, values, and the Asp-104 -- Glu generates 25% a-cuprenene and 75% trichodiene (Figure -bits wild-type lunetic pararneter~.~~However, a11 22).63,64 The X-ray crystal structure of this variant complexed ants generate aberrant cyclization products indica- with Mg2+3-PPi and (4R)-7-azabisabolenereveals a slightly temztive substrate orientations and conformations lager enclosed active site cavity that facilitates alternative {llleactive site. Thus, amino acid substitutions for product that coordinates to a catalytically obligatory Diverse product mays result from amino acid substitutions :i011, even with another residue capable of metal for ~~~-101and Arg-304, which break hydrogen bond ion (Asp-100 Glu), result in the significantly interactions in the unliganded state of the enzyme and form mod~lctarray shown in Figure 22. The crystal a double hydrogen bollded salt link in Mg2+3-PPi complexes elermination of Asp-100 - Glu trichodiene that is likely formed in the enzyme-substrate complex as trl~lexedwith PPl reveals an incomplete metal well (Arg-304 also donates a hydrogen bond to PPi, see y M< and M~Fions bound; moreover, Figure 2 The Asp- 101 -- Glu substitution results in a a lengthy 3.4 A interaction with M~:+, 5-fold decrease in and the generation of five aberrant ng to be considered inner-sphere coordina- products in addition to tri~hodiene,~~and the kg-304 - Lys mplete metal cluster results in incomplete substitution results in a 5000-fold decrease in kcatlK~,as well -iggered active site closure (Figure 23). The as the generation of at least two abenant products in addition ilve site volume is 12% greater than that of the to tri~hodiene.~~The X-ray crystal structure of Arg-304 - iurmation of the wild-type enzyme, and the Lys trichodiene synthase complexed with Mg"3-PPi and active site template allows for more confor- (4R)-7-azabisabolene shows that theoaltered hydrogen bond egrees of freedom for the flexible substrate, array shifts the PPi anion -0.7 A and weakens metal the formation of alternative cyclization products. coordination interaction^.^^ These structural changes result tion of abei~antproducts increases when Mn2+ in an enclosed active &te cavity volume 17% greater than

@#$%D1511020215HG00*&^% germacrene A iarnesyl diphosphate I

" @pi-aristolochene eudesmane cai-bocaeon Figure 24, Proposed cyclization mechanism catalyzed by epi- aristolochene synthase. Adapted with permission from ref 71. Copyright 2000 American Chemical Society.

that of the wild-type enzyme, thereby allowing for the generation of alternative cyclization products. Weakened Mg't3-PP, interactiorls implicate weakened Mg2+3-substrate diphosphate interactions in catalysis, suggesting that weaker activation of the diphosphate leaving group accounts Figure 25. Structure of epi-aristolochene synthase cornpiexed for the dramatic activity loss measured for this variant. fanlesyl hydroxyphosphonate (FHP). Tile C-ternlina! class I cycias. Clearly, even subtle perturbations in the trinuclear magne- domain (orange) contains the active site for the cycliration i-eacrio~l. sium cluster or the constellation of hydrogen bond interac- The N-terminal domain (blue) has no known catalytic f0i1ciio11 tions responsible for substrate diphosphate recognition can nevertheless adopts a ciass 11 tel-penoid cyclase fold. The bii~dij~g of substrate analogues in the C-terminal domain is accompaiiied lead to the formation of alternative cyclization products. It by the ordering of 36 residues in the N-terminal segment. and ti?c is conceivable that this strategy similarly underlies the A-C and J-K loops. These structural changes help to cap the acrjv: evolution of new terpenoid cyclases in nature. site cavity and are likely triggered by the diphosphate group of substrate farnesyl diphosphate. Reprinted with perlllissioin fio~r; Scie~zce(http:l/www.aaas.org), ref 68. Copyright 1997 hkican Associati011 for the Advancement of Science. Epi-aristolochene synthase from N. tabacunz (tobacco) catalyzes the metal-dependent cyclization of farnesyl diphos- phosphate synthase (Figure 14)" and trrckodierie syniha-,: phate to form the bicyclic hydrocarbon epi-aristolochene (Figure 21b),60 where dlphosphate/PP,7~ makes inriel-spheic (Figure 24), which comprises the first committed step in the coordination interactions with Mg, The dn er gence a, biosynthesis of the antifungal phytoalexin ~apsidiol.~~-~~The metal coordlnatron polyhedra among these tespenord cyiiaszi X-ray crystal structure of this enzyme was one of the first could reflect an evolutionary st~ategyto achreve dlve~gei~t of a sesquiterpene cyclaseh8 and revealed two a-helical cychzatmn cheinishy 111 each eiizyine acrlte site That sii~gi~ domains (Figure 25): a C-terminal catalytic domain exhibit- amino acid substrtutlons of metal-bmdlng ~esldues111 the ing the class I terpenoid synthase fold analogous to that first aspartate-rrch or NSEIDTE motifs diversify the te~peno;d observed for avian farnesyl diphosphate synthase,16 and an cyclase product array, for example, as discussed fa ti~cr~od~ N-terminal domain of uilknown function exhibiting structural ene synthase m sectlon 3.1, is consistent with tixis ncllol: homology to gluc~amylase~~and endoglucanase CelD,70 subsequently designated the class II terpenoid synthase In the epr-arrstolochene synthase rnechanisni ~ubstfdt~ The overall structure of this plant cyclase is homologous to ionization invltes attack of the C I 0-C 1 1 ,z bond z: C 1 ix llil that of (I-)-bornyl diphosphate synthase (Figure 13).47 proton elimnation at C12 to yreld the stable neubai The enzyme mechanism proceeds by initial ionization of intermediate, germacrene A (Frgure 24) Enzy~~o!oglir' the substrate diphosphate group, which is triggered by metal stud~esw~th the Tyr-520 -- Phe varrant of epi-a~~stoioche~~~ coordination interactions and hydrogen bond interactions. synthase demonstrate that ge1,nacsene A 1s formed as rhc X-ray crystal structures of epi-aristolochene synthase com- sole cyclization prod~ct.~'Although Ty-520mas I~I~I~IJ, plexes with farnesyl hydroxyphosphonde and trifluorofar- proposed as the general acld resldue responsible r'o~IJI~~~C nesyl diphosphate reveal the binding of three Mg2+ ions: nating germacrene A to form the eudesmane catro:irb (figli1~ and ~g?are figanded by Asp-301 and Asp-305 in 24) and ~tsdeletion results in the premature te~rii~nat~~flC M~Y the cyclrzatlon cascade with gelrnacrene A ihiil~an~;l the signature "aspartate-rich" sequence, and ~gris lig- Starks and colleagues68note that Asp-444 and Asp-522 nd.' anded by Asp-444, Thr-448, and Glu-452 in the NSEIDTE hydrogen bond interactrons with Tyr-520 and c~~~eei~~p;~~ motif (Figure 26). The phosphonate and diphosphate groups the possibil~tyof Asp-444 as a general acld7' 9 aaaa!o;' of the substrate analogues make coordination interactions with germacrene C synthase. 111 which an asraAtrcscjL' with ~g?and M~Tand the diphosphate group of tri- resldue 1s simlarly rrnplicated 7' This possibility IS esspee'd's fluorofarnesyl diphosphate interacts with ~g?through a rnteresting in vlew of the fact that an aspa~tc acd '" metal-bound water ~nolecule(Figure 26). This contrasts with implicated as a proton donor in pr0tonatron-depe11~~" diphosphateiPP, interactio~lswith Mg? in (+)-bornyl di- cyclases such as abietad~enesynthase (sectron 4 I j, ~cpalea'~-

@#$%D1511020215HG00*&^% Farnesyl Hyarsxyphosphonate

Mf'lrrsxo-i-amesyi pyraphosphate cy Y520- WMl& w273\0 D *

6, Bindrng of far~lesylhydroxyphosphonate (top) and tnfluoiofarnesyl dlphosphate (bottom) to the actlve slte of epi-ar~stolochene ,Metal coordinatron and hydrogen bond interactions are designated by green and blue dashed hnes, respectively Inhib~torsare I. ~thdarker- bonds than surround~ngprotein res~dues,the lsoprenoid pol-tlon of tnfluorofarnesyl dlphosphate IS sad to be character~zed electron dens~tyand is ind~catedwlth dashed bonds.6s Repnnted wlth permsslon from Sczeace (http~llwwwaaas org). lef 68 t 1997 Amerrcan Assoczatlon foi the Advancement of Science.

cj clase (section 5. I), and oxidosqualene cyclase

,Ion of germacrene A results in formation of the adesrnane cation, which then undergoes tandem I FPP I and 1,2-hydride migrations with final deproto- H rnerate epi-aristolochene (Figure 24). Clearly, the IC contour of the enzyme active site must serve Xehty template to properly chaperone reactive tes leading to epi-arisiolochene formation. The 1s lined by numerous aliphatic and aromatic -ild the side chain of Trp-273 is proposed to mterlnediates through cation-n Figure 24. Cyclization mechal~ism of ar~stolochene syna~ase erestingl~,chimeric c~clasesIn which selected Mechanlst~cdetails are summa~~zedm the text Repnnted wrth IDQ of epi-aristolochene synthase and vetisprradiene permlssron from ref 87 Copyright 2004 Amerrcan Chemicd f~omHyoscya?nus muticus are swapped generate Soc~ety of the reaction products of both enzymes, indicating actrve §ite contour of terpenoid cyclases can be dihydrofasnesyl d~phosphate,~~and a mechan~sm-basedin- accommodate alternative cyclizalion pathways hibit~r'~indicate a cychzation mechanism in which substrate from the eudesmane carbocation iIltermediate ionization is accompanied by attack of the CIO-CI I x bond both cyclases.73 hi^ work demonshraks the great at 61 w~thInversion of configuration to form the neutrai engineering diversity in terpenoid biosynthesis mtermediate 2, (9-(-)-gemacrene A; protonation at C6 and attack of the C2-C3 zbond at the resulting C.7 carbocat~on yields eudesmane cation 3, wh~chy~elds (+)-aristolochene chene Synthase 4 fnllo~vingmethyl migration, hydside transfer. and deprotonation. lxne synthase catalyzes the Mg2T-dependent (+)-Anstolochene synthases have been purlfled from P ~f famesyl &phosphate to form the bicyclic r~~uefort~~~and Aspevgzlllts le~veus,~~and both enzymes have

(+)-aristolochene 4 (Figure 271, which is a been cloned and overexpressed in E. COZL.~~79380 Both enzymes ~hlisomer of epi-aristolochene discussed in are monomei

@#$%D1511020215HG00*&^% generates appreciable quantities of (+)-aristoloch

Figure 28. Ribbon plot of (+)-aristolochene synthase from P. r~quclforti.~'The aspartate-rich motif (red) and the NSEIDTE motif (orange) flank the mouth of the active site.

crystal structure of each enzyme reveals conservation of the characteristic class I tespenoid cyclase fold.8182 The ribbon plot of (+)-aristolochene synthase from P. roqueforti in Figure 28 clearly shows the a-helical topology of the terpenoid cyclase fold and conservation of the aspartate-rich that a water molecule can be sequestered in the enzyme and NSEIDTE metal-binding motifs.*' Interestingly, Asp- site and, if firmly anchored by hydrogen bond interactjo 115, which is the first residue in the aspastate-rich motif, would not prematurely quench carbocation intermediates coordinates to a single Sm3+ ion in one of the heavy atom the cyclizatio~l~ascade.~"he X-ray crystal structure derivatives prepared in the X-ray crystal structure determi- minations of aristolochene synthase complexes with si~ nation.81This feature is reminiscent of Sm5+binding to each and intermediate analogues may illumii~atethe possib of the two aspartate-rich motifs in farnesyl diphosphate of solvent in the cyclization mechanism. synthase.16 Tlie Sm" ion is a reasonable analogue of the It is interesting that wild-type asistolochene synthase biologically relevant Mg2' ion, and each readily binds to casboxylate clusters on protein surfaces. which tend to discriminate more on the basis of ionic charge rather than ionic radius.83 The lower region of the active site of aristolochene synthase is quite I~ydrophobicin nature and its contour is defined by several aromatic and aliphatic amino acid side chains. Molecular modeling of substrate, intermediates. and product in the aristolochene synthase active site suggests that the side chains of Phe-178 and Phe-I12 are suitably oriented mechanism, result in compromised templates for ion' to stabilize the developing partial positive cl~asgeat Cl and cyclization, and it is notable that even the wil during the initial cyclization reaction, and the side chain of Trp-333 may stabilize the eudesmane cation through cation-~inte~actions.~~ Substitution of this residue with the smaller aliphatic side chains of valine or leucine results in catalytically compromised enzyme variants that essentially halt at germacrene A formation, consistent with the loss of a residue critical for the subsequent step of eudesnlane cation formation.84 3.4. Pentalenene Synthase The identification of basic residues in the enzyme active Pentalenene synthase catalyzes the Mg2+-depe site capable of performing the initial deprotonation at C12 clization of farnesyl diphosphate to form the or the find deprotonation of the eudesmane cation 3 to yield hydrocarbon pentalenene (2, Figure 30),8"."9a bbi aristolochene 4 (Figure 27) is ambiguous. Inspection of the precursor of the pentalenolactone family of antlcibi aristolochene synthase active site does not reveal any residue cyclization mechanism summarized in Figure 3 that could assist with the first deprotonation step other than clusively delineated by Cane and colleagues usi the diphosphate Ieaving group it~elf.~'Molecular modeling cifically labelled farnesyl diphosphate as a subs of the enzyme complexed with the germacrene A or allowed for the precise dissection of individual st eudesmane cation intermediates suggested that Tyr-92 could reaction sequei~ce."~~~~~~~ serve as a general acid to protonate the germacrene A Pentalenene synthase from Strept017zycesUC53 19 h intermediate and subsequently as a general base to depro- cloned, expressed in E. ~oli,~~and cry~tallized,~~ and c tonate the eudesmane cation,*l but the Tyr-32 -- Phe variant of the 42-kD recombinant wild-type enzyme yielded

@#$%D1511020215HG00*&^% l FPP 3 4 .

printed with pernlission from ref 97. Copyright 2002 American Chemical Society. decrease in kc,, and a 55-fold increase in KM,and also produces 9% of an aben-ant cyclization product, P-caryo- ph~llene.~~As noted by Seemann and ~olleagues?~the first residue in the NSE/DTE motif is aspartate instead of asparagine in the majority of plant monotespene aid sesquitespene cyclases, and the retention of aspartate or the evolution of asparagine for residue 219 in pentalenene synthase ought to support MgZi binding. Upon the initial structure determination of pentalenene syntha~e,~~Asn-219 was proposed to govern the substrate binding conformation and stabilize carbocation intermediates, but structural data subsequently acquired from the related cyclases 5-epi-aristolochene synthase," trichodiene syntha~e,~Oand (+)-bornyl diphosphate synthase4 instead suggested a conserved metal-binding function for Asn-219, Ser- 223, and 61u-227 in the NSEIDTE motif of pentalenene ~ynthase.~~Also coilsidered in the pentalenene synthase mechanism was His-309, which was proposed to mediate a possible deprotonation-reprotonatioi~sequence in the isomer- 31. Ribbon plot of pentalenene ~~nthase.~~The disordered ization of humulyl cations 3 and 4; as well as the final )-Asp-164 loop (dotted line), the aspartate-rich motif (red), deprotonation of intermediate 6 shown in Figure 30. How- NSMYI" motif (orange) flank the mouth of the active ever, subsequent site-directed mutagenesis experiments demonstrated that the His-309 - Ala, His-309 - Ser, and t class I terpenoid cycfase structures (~i~~~~3 1).96 His-309 - Phe variants of peiitalenene synthase exhibit only ier region of the enzyme active site cleft is predomi- minor increases in KM values and 3 - 17-fold decreases in drophobic in nature and contains several xomatic kc, values, with up to 22% aberrant cyclization products tic residues that define the overall contour of the formed.98 Clearly, His-309 is not required for cyclization activity, but it is required to maintain the fidelity of pentalenene cyclization. Possibly, slight alterations of the active site contour caused by His-309 substitutions compro- mise the cyclization template by conferring excessive con- formational freedom to reactive carbocation intermediates, thereby allowing formation of alternative sesquiterpene ions, hydl-ocarbons. That His-309 does not serve as a catalytically obligatory general base for pentaleneiie biosynthesis points hate toward a 12-hydride transfer instead of a deprotonatisn- reprotonation sequence to achieve the isomerization of - humulyl cations 3 and 4 in Figure 30.9x Even so, the penultimate carbocation intermediate 6 in Figure 30 must undergo a final stereospecific deprotonation of W-7re to yield pentalenene 2. Analysis of the native enzyme active site does not point to any alternative basic residues that could mediate this deprotonation step. Noting that this stereospecific step cannot be a spontaneous chenlical event and presumably must be mediated by a suitably oriented active site base, Seemann and colleaguesgs suggest three possibilities for a catalytic base: (1) enzyme-bound

@#$%D1511020215HG00*&^% water molecule(s), (2) tile carbonyl oxygen of the polypeptide DXOD DDXXD backbone, or (3) the PP, anion coproduct. The pK, values of the conjugate acid forms of all three species are sufficiently higher (greater than -4) than the pK, of a tertiary carbocation (- 10) to mediate a thermodynamically favorable deprotonation to yield a neutral hydrocarbon product. With regard to an enzyme-bound water molecule as a possible base, it is intriguing to note that a single water molecule is firmly trapped in the active site of (+)-bornyl diphosphate synthase during the monoterpene cyclization cascade,43so if a water molecule were similarly trapped in the pentalenene synthzse - active site during catalysis, it might be able to mediate the final deprotonation step. With regard to PP, as a possible base, it is intriguing that this moiety is considered as a possible base in the mechanisms of trichodiene synthase6061 and farnesyl diphosphate syntha~e.~~Clarification of these Figure 32. Panel a shows a general scheme of pla~nrdi':erpei;; synthase domain structure based on abietadiene synchase as mode]:;,,: mechanistic aspects for pentalenene synthase must await the using epi-aristolochene synthase as a template. The -9 targel;l12 X-ray crystallographic structure determinations of enzyme sequence (T.S.) is cleaved in plastids to yield a mature prore& complexes with analogues of the substrate and casbocation consisting of a -30 kD "insertional" domain of iargelg ~nicil~\~~ intermediates. function (although a pair of charged residues, Lys-86 and hrg..87, affect catalysis in the C-terminal domain), a -29 kD N-iermirIJ: domain coniaining the signature class I1 cyclase seqae!-~ce,and ,- -32 kD domain containing the signature class I cyclase sequellce Ditei-pene cyclases catalyze the cyclization of the linear DDXXD that is designated the C-terminal catalytic dornaia. 20-carbon substrate geranylgeranyl diphosphate to form a abietadiene synthase, the W-terminal and C-termina! domains variety of cyclic and polycyclic products. Although no crystal catalyze a distinct cyclization reaction. In other p!an: terpzrioii; cyclases that share this domain structure, such as taxadiene synillase. structure of a diterpene cyclase has yet been reported, amino catalytic activity is demonstrated only for the C-terminai doniaili, acid sequence similarities among plant terpenoid cyclases Reprinted with permission from ref 110. Copyright 2001 Americnii provide valuable inferences regarding structure-mechanism Chemical Society. Panel b shows a topographical projectiol: 0; relationships. Amino acid sequences of certain plant diterpene pseudomature abietadiene synthase from which tire !argeti!i; cyclases indicate the presence of both class I and class I1 sequence has been cleaved, leaving the basic Lys-86-Arg-87 pais terpenoid synthase folds containing the signature sequence ("KK') to interact with the active site in the C-terminal doinriiii ("2nd Active Site", the site of ionization-induced cyclizaiionj. Tli; motifs DDXXISiE and DXDD, respectively (Figure 32).99loo insertional domain ("Ins") links the basic pair to the It'-iei~:i:::i Unique to such plant cyclases is a -240-residue "insertional" domain ("1" Active Site", designated here as the "Central Region ~. domain at the N-terminus. Studies of abietadiene synthase the site of protonation-induced cyclization). Reprinted ~4thpermr- truncation variants indicate that residues in the insertional sion from ref 101. Copyright 2003 American Chemical Sociei!,. domain affect catalysis in the C-terminal domain.lo1 ,. Given the presence of catalytically active class I and class by a single protein, abietadrene synthase (Flgu~e351- II tespeiloid cyclase domains in the family of ditei-pene Protonation at the substrate C14 atom tr~gge~sattar:, kj ~IIL cyciases, these enzymes comprise an evolutionary bridge of ClO-Cll zbond, which triggers attack by the 26-Ci 7 sorts between the single-domain class I bacterial tespenoid bond at 611 and deprotonation at C19 to yleld the stab't cyclases such as pentalenene synthase and the double-domain bicycfic lntennedlate (+)-copalyl d~phosphate2 I" hioiilzatioli class 11 terpenoid cyclases such as the triterpene cyclases of the allylrc drphosphate ester of 2 and anti-&,' cyci~zalror' discussed in section 5. In the remainder of this section, yields the tricyclic C8-sandaracop~merenylcatloit 3a- wjJ1ck structural and mechanistic inferences regarding abietadiene then undergoes intramolecular proton transfer to ooi nl tikc synthase are discussed with regard to the distinct cyclization reactive secondary carbocation intermediate 3b' ili reactions catalyzed by its class I and class 11 cyclase domains. drrves a 1,2-methyl migratron to generate tile trrBel) A brief comparison with taxadiene synthase completes an carbocation 4+, deprotonation of which yields ableractre~~e" overview of the diterpene cyclases. or regloisomers levopimaradene 6 and neoableeaalerze Subsequent oxldat~oilof the C18 methyl gioup of 5 ylciu 4.1. Abiefiadiene Synthase (-)-abletic acid In response to a surface wound by a bark beetle. the grand Kinetic studies utilizing geranylgeranyl d1phos;~hate311. fir tree (Abies grandis) secretes oleoresin, which is a cocktail (f)-copalyl dlphosphate ~ndicatethat the two cilsl~i~ztc' of -50% volatile moiloterpene olefins (turpentine) and cl~zat~onreactions are catalyzed by two dlst~nctact]! e si~t~iis -50% ditei-pene resin acids (rosin), plus small amounts of making abletadiene synthase a b~functronalenqme S 1. certain sesq~itei-penes.'~~The volatile turpentine component dlrected rnutagenesis experrments focus~ngon tbe D~CY evaporates, leaving the hardened rosin to seal the wound. motif In the class 11 cyclase domain or the DDXXD 13c' The chemical defense mechanism is further enhanced by in the class I cyclase domain specifically abolish li insecticidal and fungicidal teipene natural products.103(-)- geranylgeranyl d~phosphatecychzat~on reactlon 01 the \- ' Abietic acid is the principal resin acid component of the copalyl d~phosphatecyclization reaction. respzctl! el, wound response in the grand fir. The hydrocarbon precursor Add~tionally,the binding of an aza analogue or gel^^'' of this resin acid is (-f-abieta-7(8),13(14)-diene, or simply geranyl diphosphate, 15-aza-GGPP,11' preferentla:l\~~l~ll'~'' abietadiene. the cycllzation of geranylgeranyl d~phosphatebut "' Abietadiene is generated by the cyclization of geranyl- cyclization of (f)-copalyl drphosphate.""xe (-)-cOi?L ' geranyl diphosphateiO%ntwo consecutive reactions catalyzed d~phosphateis detected HI steady-state assays, coo,~t~~~~5'i1'

@#$%D1511020215HG00*&^% class II cyclase active site at the C-terminal domain.lol Interestingly, (+)-bornyl diphosphate synthase also contains a pair of charged residues, Arg-55 and kg-56, in its N-terminal domain, but it does not coi~tain an entire "insertional" domain comparable to that of the plant diterpene cyclases. Arg-56 of (+)-bo~xyldiphosphate syntliase donates a hydrogen bond to the carbonyl of Asp-355 in the aspartafe- rich motif in the coinplex with (+)-bornyl diphosphate or ternary complexes with PPi and analogues of carbocation~ - intermediates, thereby facilitating the "capping" of the active - -. siCiy the R-tm1;i,na~polypeptide?3If Arg-55 and Arg-50 of (f)-bornyl diphosphate synthase co~~espondto Lys-86 and kg-87 of abietadiene synthase, it is iilteresfng to speculate that these abietadiene synthase residues may likewise interact with the diphosphate group of (+)-copalyl dipliosphate, or at least they may be involved in facilitating the contact between the insertional domain and the class 1 cyclase domain, which could possibly serve to cap the active site cavity.lo1 The X-ray crystal structure determination of abietadiene synthase complexed with substrate a~ialogues should help to pinpoint features of substrate recognitiol~and catalysis in both active sites of this novel bifunctional cyclase. 42. Taxadiene Synthase Paclitaxel (Taxol) is a taxane diterpene isolated from the bark of the Pacific yew (Taxus br-e~ifolia)'~~that exhibits potent antitumor properties by s~abilizingmicrotr?bules and arresting the cell cy~le."~-"~Intriguingly, recent reports show that paclitaxel may also be useful iii treating neuro- degenerative diseases such as Alzheimer's disease by compensating for tlie loss of function of the microtubule- binding protein tau.H8p120Taxadiene synthase catalyzes the first compnitted step of paclitaxel biosynthesis through the metal-dependent cyclization of geranylgeranyl diphosphate iffusion transfer of (+)-copalyl diphosphate out of to form taxa-4(5),1 l(12)-diene (Figure 34). site of the class II cyclase domain and into the Taxadiene synthase has been isolated and purified from F of the class I cycIase domain, so there is no the Pacific yew,121 and the full-length, 862-residue enzyme for a more complex substrate channeling mecha- (98 la)has been cloned and expressed in Eschel-iclzia coli.12' The amino acid sequence displays 46% identity with that of abietadiene ~yntfiase,~~%otaxadiene syntl~aseshares the general domain structure outlined in Figure 32a. The preparation of a truncation variant in which 60 residues of the plastidiai targeting sequence are deleted from the N-terminus results in a "'pseudomature" form of the eazymne that facilitates overexpression in Esclzerichia culi, purifica- tion, and assay.123This recombinant enzyme generates taxa- 4(5),1 l(12)-diene as the major cyclization product /94%), but minor amounts of side products, taxa-4(20),1 I(12)-diene (-5%) and verticillene (--I%), are also detected. Given that taxadiene synthase contains both class I and class II cyclase folds, as indicated by its significant amino acid sequence identity with abietadiene synthase, tlie first question regarding structure-mechanism relationships focuses on which domain(s) catalyze the cyclization cascade. Amino acid sequence alignments.of taxadiene synthase and abietadiene synthase reveal that the signature DXDD motif of the class I1 cyclase domain of abietadiene synthase appears niscent of recent structural studies of site- as DLNT in taxadiene synthase; since the central aspartate of this domain is typically the general acid that triggers the protonation-induced cyclization cascade of abietadiene syn- thasel l2 and the triterpene cyclases (section 5), and since the f abietadiene synthase truncation variants central aspartate is absent in taxadiene synthase, it appears ir of charged residues in the N-terminal that the class I1 cyclase domain is not equipped for and Arg-87, in the catalytic activity of the protonation-induced carbocation formation. Instead: the entise

@#$%D1511020215HG00*&^% 2, GGPP 3 4

Figure 34. The cyclization of gerailylgeranyl diphosphate (GGPP, 2) catalyzed by taxadiene synthase requires Mg': to trigger t!ie ii of the pyrophosphate leaving group (OPP). The cyclization cascade tesminates with.the formation of taxa-4(5), 1 l(12)-diene 6, which ti further biosynthetic derivatization in the Pacific yew to yield paclitaxel (Taxol, I). Reprinted from ref 126, Copyright (2000),with peri:lissi fsom Elsevier.

cyclization reaction occurs in tile C-terminal class I cyclase squalene oxide to generate a variety domain and is triggered by the Mg2+-dependent departure Initial carbocation formation is achi of the geranygeranyl diphosphate leaving group. Studies wit11 a carbon-carbon double bond in s isotopically labelled substrates indicate that the CWzOPP protonation and ring opening in squalene oxid group and the C14-C15 and CIO-Cll x bonds of gesanyl- aspartate in the signature DXDD amino acid sequence m geranyl diphosphate are favorably aligned for the possibly is implicated as the requisite proton donor. Interest' concested alkylation and x-cyclization to form the verticillyl squalene derives from two ntolecules of farnesyl diph~s carbocation intermediate 3 (Figure 34) and confirm that the connected in "head-to-head" fashion by squalene synt Cl atom of the substrate undergoes inversion of configuration the structure of which reveals topological similarities upon A ring f~rmation.'~~-~~~ class I terpenoid ~ynthases.'~ Additional studies with specifically deuterated substrates Despite the size of their substrates, the tr indicate an intramolecular proton transfer of the Cl I proton can achieve the same degree of struct~valand s

to the re face of G7 to generate tertiary carbocation precision in catalysis as previously noted for class 1 [el 1 intermediate 4 (Figure 34).i24126This proton transfer is cyclases. The X-ray crystal structures of t somewhat unusual in that a x-bond of the substrate, rather cyclases, squalene-hopene cycla~e~'~~'~~ than an enzyme-bound residue, serves as a "general base" cyclase (also known as lanosterol sy to accept the C11 proton. This step is analogous to the a critical foundation for understandin intramolecular proton transfer proposed for the sandara- relationships in this enzyme family. Give copimerenyl cation in the abietadiene synthase mechanism and outstanding review of triterpe~lecyclization (3a+ -- 3b+ in Figure 33). When 6-fluoro-geranylgeranyl has been presented,1° here I focus primaril dipkosphatc is used as a substrate with taxadiene synthase, aspects of triterpene cyclase stmcture-

the intramolecular Cl l -+ C7 proton transfer is slower due ships discerned from expektnentally determine to the electron-withdrawing effects of fluorine and B/C ring structures reported within the past two years. closure is b10cked.l~~The resultant isolation and character- ization of partially cyclized fluoroverticillenes shows that the stereochemistry at C11 of the verticillenyi carbocation intermediate is 1 lR.12' Squalene-hopene cyclase catalyzes the Transannular B/C ring closure of carbocation intermediate squalene to form the pentacyclic hydrocarbon 4 leads to formation of the taxenyl carbocation 5 (Figure 35). Hopene is a biosynthetic precursor of the 34). Enzj~mologicalstudies utilizing specifically deuterated hopanoids, which modulate membrane flui geranylgeranyl diphosphate indicate that isomeric side crystal structure of squalene-hopene products derive from this intermediate;'" elimi~~ationof the clobacillus acidocaldavius reveals a di W5P proton terminates the cyclization cascade to form taxa- each moilomer contains two domains, and 4(5),11(12)-diene 6 (Figure 3).lZ6Although the role of the adopts a double a-ban-el fold. The two dom enzyme in catalyzing this reaction is not clearly defined (e.g., so as to enclose a central hydrophobic acti does an enzyme residue mediate the final deprotonation of (Figure 36). the taxenyl cation?), the future X-ray crystallographic Squaiene-hopene cyclase is a monotopic me!< structure determination of taxadiene synthase will allow such protein, meaning that it penetrates but does not ~01114 detailed stl-ucture-mechanism relationships to be conclu- pass through the bacterial membrane. Substrate sq.2 sively e~tab1ished.l~~ dissolved in the membrane, enters the cyciase actii e though a hydrophobic channel open to t surface. A nonpolar "plateau" flanks the channel .enr in the vicinity of a-helix 8, and this likely The h-iterpene cyclases are designated as class 11 tespenoid monotopic membrane'association motif (Figu cyclases because their double a-barrel fold is topoiogically As with the class I terpenoid cyclases, the acti\.e sit distinct from the characteristic a-helical fold of a class I squalene-hopene cyclase is very hydrophobic in riaillre terpenoid cycfa~e.~Tritelpene cyclases catalyze the cycliza- a variety of aromatic and aliphatic amino acid side tion of the linear 30-carbon isoprene substrates squalene or define a precise active site contour that serve

@#$%D1511020215HG00*&^% ar-e essentially inactive.134Similar amino acid substitutions of Asp-377 result in virtually inactive enzyme variants: pr-esumably due to the disruption of the Asp-346 environ- ment. Consistent with these mutagenesis results, the side chain of Asp-376 is oriented toward the W-63 bond of 2-azasqualene in Figure 38, which corresponds to the face of the C2-C3 3 bond of squalene (Figure 39).j3? On the basis of the crystal structure of the complex between squalene-hopene cyclase and the poiyisoprenoid substgate anahgye 2-azasquaiene (Figures 38 and 39), a. 29 -. -.. Wopene mechanism for hopene formation is proposed13' that i~lvolves =e 35. Squalene-hopene cyclase reaction. Various data low-barrier Markovnikov-type cation-olefin additions to te that general acid BI:H is Asp-376; which protonates form the six-membered A and B rings in the chair confomza- ene at C3 and triggers the cyclization cascade. The crystal tion, with a tes-tiary carbocation intermediate at C10. The cture reveals no specific residue that could serve as general binding conformation of 2-azasqualene suggests that although e B:: for the final deprotonation step at C29. This could account C10 e generation of 10% hopan-22-01 as a side product resulting it is possible that the carbocation could react with the quenching of the final C22 carbocation by solvent. Reprinted C14-C15 x-bond in Marliovnikov fashion to yield a five- permission from Science (http://www.aaas.org), ref 129. membered C ring intermediate, it appears more likely that a ight 1997 American Association for the Advancement of six-membered C-ring is formed directly by anti-Markavnilov nce reaction of the C14-C15 JT bond with the CIO carbocation in concert with Markovnikov reaction of the incipient force the catalytically productive conformation of the secondary C14 carbocation with the C18-C19 double bond e. flexible polyisoprene substrate. The recent X-ray to yield a five-membered D ring with a resulting tertiary 'lographic structure determination of the complex with carbocation ion at C19. This intermediate is designated the areactive substrate analogue 2-azasqualene provides 6-6-6-5 tetracycle with rings A-C adopting the chair- gant illustration of this template function (Figure 38), chair-chair conformation of hopene. Approach of the C22- the substrate analogue binds with a conformation very C23 JZ bond toward the C19 carbocation appears to be to that required for the chair confornlations of hopene sterically hindered, leading Reines-t and colleagues to suggest that the 6-6-6-5 carbocation is a relatively long-lived sho1-t amino acid sequence DXDD/E has been identi- intermediate.r32That derivatives of the 6-6-6-5 carbo- s a consensus motif in squalene cy~lases,~~~:'~~and site- cation are observed as minor byproducts in the squalene- d mutagenesis has been used to probe the catalytic hopene cyclase reaction135and as significant byprod.ucts !I of aspartate residues in the squalene cyclase reaction. generated by site-specific variants of squalene-hopene r~tralacidic residue; Asp-376: is the likely general acid cyclase'" is consistent with a 6-6-6-5 intermediate."" sible for protonating the C3 atom of the squalene Subsequent D-ring expansion and Markovnikov E-ring ate. The Asp-376 - Glu variant retains 10.3% relative formation yieIds the tertiary hopenyt carbocation, which tic activity compared with the wild-type enzyme, undergoes proton elimination to yield hopene. Detailed as the Asp-376 -- Gly and Asp-376 - Arg variants aspects of the chemical mechanisms leading to the formation

tlomaii~2 Dolt~ain2 of the squalene-hopene cyclase monomer. Amino (N)and carboxyl (C) terminal residues are located in domain 1. a-helices of the two &a-barrel domains are yellow and red, respectively. A small p-sheet (green) comprises one wall site cavity (L), which is accessed through the hydropl~obicchannel entrance (E). Loop segments containing characteristic an motifs are magenta; a-helix 8 in domain 2 (white) anchors the enzyme in the membrane. Reprinted with permission ww.aaas.org), ref 129. Copyright 1997 American Association for theAdvancement of Science.

@#$%D1511020215HG00*&^% tion in the lower region of the active site cavity tritelpene cyclases thus allows for divergent biovnrh reactions. 129 5-2, Oxidosqualene Cyclase Oxidosqualene cyclase catalyzes the exclus of oxidosqualene to forn~lanosterol, a critica biosynthesis of cholesterol (Figuse 40). Human o cyclase functions as a monomer and exhibits --aid sw;teli;ce identity with squalene-hopene c Oxidosqualene cyclases usually contain only th aspartate corresponding to the DXDD m squalene cycIases: in human oxidosqualene cyc 8.e corresponds to Asp-455, and in oxidosqualene cyclase entrance Sacchal-onzyces cer-euisiae this co~respondsto Asp-4.56. directed nlutagenesis with the enzyme from S. cel-eui:;~: the binding of irreversible inhibitors implicate Asp-4 Figure 37. Model of dimeric squalene-hopene cyclase in the the general acid that protonates the substi-ate epoxide membrane. The hydrophobic active sites and channel entrances are to initiate the cyclization indicated, and general acid Asp-376, which initiates the cyclization The X-ray crystal structure of human oxidosqu cascade by protonating C3 of squalene, is marked as a red dot near the "top" of each active site. Recent X-ray crystallographic studies cyclase reveals dual double a-barrel domains conr~ected indicate that the outer channel wall, including Phe-434 (black dot), loops and a smaller P-sheet structure with identical to is mobile (red arrows). Such mobility presumably facilitates to that observed in squalene-hopene cyclase (Figure substrate binding and product dissociation from the enzyme active As proposed for squalene-hopene ~yclase,'~~a hgldl-op site by allowing the channel to open fully. Reprinted from ref 132, plateau Ranking a-helix 8 likely serves as the rnoliot Copyright (2004), with permission from Elsevier. membrane insertion motif. The binding of substrate oxi of hopene as well as alternative cyclization products have squalene and the release of product lanosterol occurs :b been reviewed by Wendt and colleag~es.'~ a hydrophobic channel adjacent to a-helix 8 that con When the structure of squalene-hopene cyclase was the active site cavity to the membrane surface. Howe initially repo1~ed.l~~it was noted that there is more conserva- secorzd active site channel is observed in oxidos tion of amino acid residues in the upper region of the active cyclase: a polar, solvent-accessible channel leads to t! site cavity (i.e.. the polar, Asp-376 region) than in the lower (i.e., the Asp-455 region) of the active site cavity, a region of the active site as squalene-hopene cyclase is side chain of CIu-459 is located at the midpoint compared with other triterpene cyclases, suggesting that the channel (Figure 41b). first reaction step common to squalene and oxidosqualene The structure of human oxidosqualene cyclase compi cyclases, substrate protonation, occurs in the conserved with product lanosterol interpreted in view of enzymoIo region of the cavity. The divergence of amino acid composi- studies allows for the conclusive determinatio~lof ac

Figure 38. Electron density map showing the precise binding conforination of the polyisoprenoid substrate analogue inhibitor 2-aza in the active site of squalene-hopme cyclase. The inhibitor conformation corresponds to the productive squalene confom~atlor for hopene biosynthesis, thereby exemplifying the precise template function of the tei-penoid sj7llthase active site. Reprinted from Copyright (2004), with permission from Elsevier.

@#$%D1511020215HG00*&^% 4, Model of squalene (green) based on the experime~ltallydetermined binding conformation of the nonreactive substrate analogue a!ene (thin dark bonds). Dotted green lines indicate the first four carbocation addition reactions at C2, C6, CIO, C14-C15, and yield the 6-6-6-5 tetracycle. Reprinted from ref 132, Copyright (2004), with permission from Elsevier.

cationic cyclization cascade.'" a reactive carbocation, a hydrogen bond with Tyr-503 appears , the side chain of Asp-455 to be sufficient to prevent His-232 from adopting a confor- sterol hydroxyl group, consis- mation that would allow it to react with a C14 carbocation. in the proposed role of Asp-455 as the general acid The closure of the D-ring yields the protosterol C20 cation, lates a proton to the epoxide oxygen of the substrate which is subsequently converted into lanosterol by tandem te the cyclization cascade (Figure 42a,b).'38The side 1,2-hydride transfer and 1,2-methyl migration reactions (Figure 40). Thoma and colleagues1" suggest that the side chain of His-232, an essential and strictly conserved residue among the oxidosqualene ~yclases,'~~is the only possible candidate for the base that accepts the C9 proton ultimately eliminated to terminate the cyclizatioll cascade (Figure 40). s suggest that reprotonation of Asp-455 could be However, inspection of the active site also reveals that Tyln- by bulk solvent though a hydrogen bonded solvent 503 (also conserved among the oxidosqualene cyclases) could with 61u-459 in the polar channel leading to Asp- possibly accept this proton, and it is closer and better-oriented with regard to the C9 proton that is ultimately eliminated to form lanosterol (Figure 42). Wow that a thl-ee-dimensional crystal structure of oxidosqualene cyclase is available, the catalytic functions of Tyr-503 and other active site residues can be probed in further detail to clarify structure-rnecha- ra~ti0ns.l~~Specifically, Trp-387, Phe- nism relationships for this triterpene cyclase. tabilize tertiay carbocation intesmedi- 42b). Additionally, Tyr-98 6. Ant%ody Cafajysjs of Cgtjonic Cyclization vity in such a manner that Reactions ilize the substrate in the unfavorable tion required for lanosterol B-ring formation. To date, several catalytic antibodies have been generated the class I terpenoid cyclases, active site that catalyze cationic cyclization reaction^.'"^^^^ Especially notable among these novel biological catalysts is antibody 19A4, which catalyzes the tandem cationic cyclization of a linear substrate to form a trans-decalin sing system analogous to that of the steroid A and B rings (Figure 43).'" In comparison, antibody 4C6 catalyzes a slightly simpler reaction, the cyclization of a linear substrate to form a s only briefly recounted here in cyclohexane ring system (Figure 44). A key biosynthet~c -ray crystal structure of the enzyme. The anti- advantage of catalytic antibody cyclases is that they are not limited to the handful of naturally occurring isoprenoid polyene substrates illustrated in Figure 1. Therefore, the potential product diversity of antibody cyclases is even greater than that of the naturally occurring te~penoidcyclases. Although the characteristic /%fold of the immunoglobulin Fab fragment is topologically distinct from the a-folds of

@#$%D1511020215HG00*&^% Figure 40. Oxidosqualene cyclase, also known as lanosterol synthase, catalyzes the cyclization of (S)-2.3-oxidosqualeneto form lanosterol, a key intermediate in cholesterol biosynthesis. The initial substrate chair-boat-chair conformation, proposed confonnations following A-. B-, and C-ring closure, and rearrangement of the protosterol cation via I .2-hydride and 1,2-methyl transfers are indicated. Reprinted with periliission from Nature (http:l/ www.nature.com), ref 131. Copyright 2004 Nature Publishing Group. Figure 41. Panel a shows a ribboil diagram of human the naturally occurring terpenoid cyclases, the X-ray crystal squalene cyclase. Inner helices of each &a-ban-el are yelio the inhibitor Ro 48-807 1 (black) indicates the location of the a structures of catalytic antibody cyclases 19A4 and 4C6 reveal site cavity. Panel b shows a model of human oxidosqualei~ec ,c important features in the antibody combining sites indicating associated with a membrane leaflet (polar and nonpola I convergent structure-mechanism relationships with the the membrane are colored light blue and light yellow. re terpenoid cyclases for the management and manipulation of Internal surfaces are colored as follows: blue, po reactive carbocation intermediates.14' 14* Active site features negative: cyan, hydrogen bond donor; magenta, other po shared by the "unnaturally-evolved" antibody cyclases and detergent molecules or lipid fragments observed in the "naturally-evolved" terpenoid cyclases include (1) a structure (blue and black stick figures) cluster around the insertion region. Reprinted with pernlission from Nat~:i- hydrophobic active site cavity capable of sequestering the www.nature.com), ref 131. Copyright 2004 Natui-e Pu substrate from bulk solvent, (2) a precisely formed active Group. site contour, that is, a three-dimensional template, that selects for the productive col~formation(and selects against unpro- 6.4. Catalytic Antibody 1984 ductive conformations) of the flexible polyene substrate, (3) a "carbocation trigger", that is, polar residue(s) that trigger The X-ray crystal stsucture of the Fab fragment of ca initial carbocation formation once the substrate is bound in antibody 19A4 complexed with the bicyclic hapten 5 s a productive conformation, and (4) aromatic residues that in Figure 43 reveals that the hapten is buried deep1y may stabilize high-energy carbocation intermediates through the antibody combining site and makes numerouc val cation-x interactions. In the remainder of this section. Waals contacts with aromatic and aliphatic resi structure-mechanism relationships for catalytic antibody mediate a highly complementary fit (Figure 451.'" cyclases 19A4 and 4C6 are summarized to illustrate these precise contour of the antibody combining site serv convergent features in the catalysis of carbocation cyclization three-dimensional template that binds the flexible p cascades. substrate in the productive chair-chair confomatiop re

@#$%D1511020215HG00*&^% @#$%D1511020215HG00*&^% - - H ' solvolysis

\ LGM = CH2C0NHC6H4NHCO(CH2),C4 - .\ 5 4a,b Figure 43. Catalytic antibody 19A4 catalyzes the formation of traits-decalin regioisomers 2a-c as the major cyclization producrs fro substrate 1; premature quenching of the first carbocation intermediate yields olefins 3a-c or alcohols 4a,b.14 Kinetic chxacterira~j based on the rate of sulfonic acid release yields kc,, = 0.021 min-' and KM = 320 pM; for solvolysis, kUncat= 9.2 x 1W6minpi; so ~h acceleration for leaving group departure is 2.3 x lo3. Bicyclic hapten 5 was used to elicit the antibody and partially mimics the prodnct chair-chair conformation required for cyclization to form the tmizs-decalin; additionally, the leaving group mimic (LGM)lN-oxide moi was incorporated into the hapten design to elicit functional groups in the antibody combining site that might facilitate leaving group i' departure. Q Q Me- Si,-Me Me-si-Me + LG

Leavmg Group Mimic Plus

Figure 44. Cationic monocyclization reaction catalyzed by antibody 4C6 affords products ti-ans-2-(dlmethylphenylsily1)-cyclohexanol 3 (98%) and cyclohexene 4 (2%).i40J48Hapten 5 was used to elicit the catalytic antibody. Reprinted from ref 148, Copyright (2003), 1~1th permission from Elsevier.

antibody cyclase would be the initiation and stabilization of antibody may not only incorporate structure features a1 the first carbocation intermediate; accordingly, the N-oxide moiety of hapten 5 would be appropriately located to elicit complementary residues in the antibody combining site that could trigger leaving group departure and stabilize the first carbocation i~~termediate.'~~The structure of the 19A4- The proposed cyclization mechanism catalyzed in hapten 5 complex reveals a hydrogen bond between the active site of antibody 19A4 is summaxized in Figure 40. negatively charged oxygen atom of the AT-oxide moiety and In concert with leaving group departure triggered by the side chain carboxamide group of Asn-H35A, thereby suggesting that this residue helps trigger the departure of the sulfonate leaving group in catalysis (Figure 45b).147 of the trans-decalin with the tertiary C5 carbocation Additionally, the positively charged nitrogen atom of the by cation-z interactions with ~rp-~91and Tyr-L N-oxide moiety interacts predominantly with the z-electron 46b). The generation of minor olefin side products 38 clouds of Trp-L91 and Tyr-L96. Since this nitrogen atom and alcohol side products 4a,b (Figure 43)144is coii~i conesponds to the substrate Cl atom (Figure 43), which with formation of a carbocation intermediate at C5.TI develops partial positive charge during leaving group de- carbocation is subsequently attacked by the C9-ClO z parture, it appears that the incorporation of the N-oxide to yield the six-membered B-ring of the trans-decalin (F moiety in hapten 5 successfully elicited residues capable of 46c). The resulting carbocation intermediate at stabilizing the developing partial positive charge at Cl stabilized by cation-z interaction (Figure 46a). This feature, too, may facilitate leaving group departure in catalysis. Thus, a hapten used to elicit a catalytic

@#$%D1511020215HG00*&^% anel a sho~vsthe backbone Ca trace of the variable region of antibody BA5-19A4 con~plexedwith hapten 5 (Figure 43). In electron density map of hapten 5 bound to antibody HA5- 19A4 reveals that numerous aromatic residues surround the hapten ly complementar)~surface, and this surface contour must accordingly be highly complenlentary for the corresponding tandem ction cascade yielding t~alz,r-decalinregioisomers 2a-c shown in Figure 43 (for clarity, only a portion of the ieaving group is shown). A hydrogen bond between the N4-0- n~oiet)!and Asn-K35A suggests that this residue triggers leaving group talysis. Reprinted with permission from ref 147. Copyright 1999 Wiley-VCH.

11 sire of developing partial or full positive charge on the ~necfianisticproposal in which the active site stabilizes the siraie-C1, C5. and C9-is potentially stabilized by two binding of substrate in a quasi-chair-like conformation to ion-- 7 interactions. accommodate the expected transition state structure with the inir-iation of the cyclization cascade is achieved by sulfonate leaving group in a pseudoequatorial position elimination to yield trans-decalin regioiso~ners2a-c (Figrrre 48).'40.'48The aroinadc rings of both Tyr-L96 and re 4Sd). Although the oxirane moiety was incorporated Tyr-If50 are oriented such that they could donate hydrogeri design of hapten 5 to elicit a polar residue that could bonds to the sulfonate ester oxygen and thereby trigger 1:- a specific ter~i~inationreaction for the cyclization leaving group departure. The aromatic rings of Tyr-L96, TIT- -. ti-uizs-decaiins 2a-c are generated with product H97, and Pbe-L32 could stabilize the developing partial rei'iecting their expected thermodynamic stabilities.'14 positive charge at Cl that accompanies leaving group e crystal structure of the HA5- 19A4 complex with hapten departure in concert with nucleophilic attack by the C5-Ct vea!~that there is 110 catalytic base appropriately .T bond, and Tyr-MSO and Tq-H97 may stabilize tile C5 fied in the antibody combining sire to direct the carbocatio~lintermediate through cation-z interactions. The ;einisti-y of the final elimination step to yield one reaction is terminated primarily by addition of a water srl e eIimination product.'" Thus, in this system the molecule to the C5 caxbocation to form rmrzs-2-(dimethyl- r r~zoietyis not as successful as the AT-oxide rnoiety in pheny1silyl)-cyclohexanol, although minor amounts of cy- sit-and-switch" strategy aimed at eliciting a comple- clohexene are formed by elimination of the dimetlzyIphen- ,y functional group in the antibody combining site.'@ ylsilyl group (Figure 5).I" A detailed structural comparison of catalytic antibodies . Ga:ahytie Antibody 466 19A4 and 466 is presented by Zhu and colleag~es.'~"n e cacionic monocyclization reaction catalyzed by anti- general, the combining site of 19A4 extends more deeply "106 is shown in Figure 44.'" Here, too, an N-oxide into the interface between the variable regions of the heavy y \);as ilicorporated into the hapten as part of a "bait- and light chains than that for 4C6. It is also notable that the "iiicii" strategy:fio.fiithe negatively charged oxygen molecular recognition of the N-oxide moiety of the hapten of A'--oxidehapten 5 in Figure 44 was intended to elicit differs for each catalytic antibody: 19A4 utilizes an aspar- es {.hat could potentially stabilize the developing agine side chain, whereas 3C6 utilizes a tyrosine side chain, charge on the sulfoliate leaving group, and the to donate a hydrogen bond to the negatively charged oxygen y charged nitrogen atom was intended to elicit of the N-oxide moiety. Finally, numerous aro~llaticresidues s capable of stabilizing the developing partial positive are found in each antibody combining site and may stabilize e at rlle corresponding substrate C1 atom as well as high-energy carbocation intermediates through cation--ir lbseijuently for~iiedC5 carbocation. interactions. rey crystal st~uctul-eof the 4C6 Fab complexed with ~liustl-atedin Figure 44- reveals that the phenolic Z Cone!uding Remarks cC Tyr-L9G donates a hydrogen bond to the oxygen A total of nine X-ray cr-ystal structures of terpenoid N-oxide moiety (Figure 47),IJ8thereby confirm- cyclases and catalytic antibody cyclases have been reported e s:iccessful "bait-and-switch" strategy underlying in the past eight years, and it is instructive to compare these As expected, the three-dimensional contour enzymes to understand the structural basis for their biosyn- afltlilody combining site is quite complementary in thetic diversity. In terms of their structurat biology, the the hapten. The binding pocket is primarily naturally occurring terpenoid synthases adopt one of two c in nature and contains several aromatic residues topologically distinct a-helical folds, designated the class I I;zIIy participate in catalysis (Figure 47). Structural and class 11 terpenoid synthase folds.6 The class 1 tapenoid of the 4G6 Fab-hapten complex leads to a cyclase fold achieves initial carbocation formation by

@#$%D1511020215HG00*&^% Figure 46. Proposed cyclization mechanism of catalytic antibody HAS-19A4 based on enzymological and X-ray crystal 26; 1 analysi~.'~.'"~'~~Structural chemical aspects of this mechanism are discussed in the text. Reprinted with permission from ref 149. 2000 Chiana M. Paschall. unifc substrate ionization, and the class I1 terpenoid cyclase fold are formed by single a-barrel structures homologo achieves initial carbocation formation by substrate protona- farnesyl diphosphate synthase (Figure 3);16 the active tion. of a class I1 diterpene cyclase, the N-terminal domain of In class I and class TI terpenoid cyclases, amino acid side bifunctional cyclase abietadiehe synthase, is formed 5 chains critical for catalysis are located on or adjacent to single-domain a-barrel structure based on amino a-helices that form a barrel-type superstructure in which sequence analysis (Figure 32);I0l and two a-barrel do active site is nested. The active sites of class I terpenoid associate in head-to-head fashion to form the aciiv cyclases (monoterpene, sesquiterpene, and diterpene cyclases) cavity of a triterpene cyclase with substrate access t1.i

@#$%D1511020215HG00*&^% iinemlcal Reviews, 2006; Vol. 106, No. 8 3439

are 4'7. Panel a shows the shape complemeiltarity between the combining site of catalytic antibody 4C6 and hapren 5 illustrated ii~ re 44. Panel b presents a stereoview of the 4C6-hapten 5 complex showing antibody combining site resid~lesthat inay participate in ysis. Reprinted from ref 148, Copyrigl-rt (2003), wit11 permission froin Elsevier. phak synthase (Figuses 5 and 6) and (+)-bornyl diphosphate synthase (Figure 14); it coordinates to ~g: and only poorly, if at all, to hfg? in the active site of epi-arisro- lochene synthase (Figure 26): and it does not coordinate to any metal ions in the active site of trichodiene synthase (Figure 21b). Thus, the first aspartate in the aspartate-rich motif has retained a cornmoll metal coordination function in the evolution of class I terpenoid synthases of ltnown structure, whereas the metal coordination geometry and function of the third aspartate is more divergent. Divergent function is also implied by divergent amino acid sequence

+ Q as well, for example, the third aspartate in the DDXXD motif Me- st-Me appears as glutamate in certain terpenoid cyclases suclz as I OH aristolochene synthase (section 3.3), and two additional residues are inserted in one of the DDXXD motifs to yield a DDXXXXD motif in farnesyl diphosphate synthase fi-om E. c01i.'~ re 48. Proposed cyclization mechanism of catalytic alltibody The second metal-binding motif of the terpenoid c)~clase, ased on enzymological and X-ray crystallographic analy- (L,V)(V,L,A)(N,D)D(L,I.V)X(S,T)XXXE(the "NSE/DTEW '""tructural chemical aspects of this mechanism are ssed in the text. Reprinted from ref 148, Copyrigilt (2003), motif; boldface residues form a complete ~gg-binding permission from Elsevier. site) appears to have diverged from the second aspartate- rich motif of farnesyl diphosphate ~ynthase.~'.~~The NSE/ moui tunnel at the interdomain interface. The generation DTE motif uniformly chelates a single metal ion, ~g2,in ody catalysts that cyclize isoprenoid-like substrates (+)-bornyl diphosphate synthase (Figure 14), trichodiene :rates that carbocation cycIization cascades are readily synthase (Figure 21b), and epi-aristolocbene synthase (Figure ed within /$-sheet frameworks as well, so long as there 26). This motif therefore distinguishes the amino acid table mechanism for triggering carbocation formatio~l sequence of a terpenoid cyclase from that of a chain- the substrate is bound in a productive confol-mation. elongatiilg terpenoid synthase. "kspai-tate-rich" motif17-l9 DDXXD/E and the "NSE/ X-ray crystal strustures of class I terpenoid cyclase ~xotif~'~~~(N/D)DXX(S/T)XXXE are located on op- complexes with PPi, substrate analogues, and product reveal sides of the active site cleft of a class I terpenoid that the complete triiluclear magnesium cluster is stabilized (e.g., see Figure 131, and these motifs signal the by the substrate diphosphate group, the product PPi anion, of a trinuclear magnesium cluster that triggers or both, which make numerorrs metal coordination and e ionization and initial carbocation formation. Crystal hydrogen bond interactions (e.g., see Figures 5, 6, f 4, 21b, res of farnesyl diphosphate synthase (Figures 5 and and 26). These interactions trigger structural changes in my1 diphosphate synthase (Figure 14), trichodiene helices and loops Ranking the mouth of the active site such e (Figure 21b), and epi-aristolochene synthase (Figure that the active site cavity is closed and sequestered from bulk eal that the first aspartate in the aspartate-rich motif solvent. This ensrues that the productive active site contour, rnly coordinates to two metal ions, Mg,2+ and Mgc21- , the template for the cyclization cascade, is formed only after n3.i-3;n-bidentatecoordination stereochemistry. The the substrate is fully bound and ready for ionization. aspartate in this motif does not male any direct Significantly, even subtle changes in the metal coordina- ons with metal ions, but it does appear to engage in tion polyhedra dramatically alter cyclization product diver- portant functions in some enzymes, for example, sity, even while sustaining 'appreciable catalytic activity as '01 -kg-204 salt link stabilizes the closed active described for site-specific variants of trichodiene ~ynthase.~'.~' mation of trichodiene synthase (Figure 21 d). The This suggests that one evolutionary strategy for teqene partate in this motif bridges and ~~g+product diversity may be the introduction of amino acid a single carboxylate oxygen with syn;aizti coordina- substitutions in the. metal coordination polyhedra, or residues eochemistry in the active sites of farnesyl diphos- that abut the trinuclear magnesium cluster, which can then

@#$%D1511020215HG00*&^% result in altered cyclization reaction. That the generation of class II cyclases such as squalene-hopene cyclas- alternative cyclization products increases when Mn2' is oxidosqualene cyclase, the active site coiltour plays substituted for Mg2' in assays with Asp-100 -- Glu role as a template throughout the cyclization cascade. trichodiene synthase clearly demonstrates that the metal template must not only guide the folding of the coordination polyhedra and substrate diphosphatePP, inter- isoprenoid substrate but also guide the folding actions play a crucial role in governing cyclization product subsequently formed intermediates that can differ si cantly from the substrate in terms of size, shape, conf In contrast with the class I terpenoid cyclases, the class I1 tibnal flexibility, and charge distribution. Thus, the a terpenoid cyclases trigger initial carbocation formation by site co~ltoui-of a high-fidelity terpenoid cyclase is optin substrate protonation rather than ionization. Class II teipenoid - for complementadty to multiple structures along the rea cyclases contain the DXDDW in which the .-~oo~&Eziie--- of -c1icii.tation (such complementarity central aspartic acid serves as a proton donor to either a conceivably require flexibility in the ternplate its carbon-carbon double bond (e.g., as in the N-terminal contrast, the active site contour of a terpenoid cvcl domain of abietadiene synthase (section 4.1) or squalene- generates multiple products is more pronrisc hopene ~yclase(section 5.1)) or the epoxide oxygen of complementarity to one or more structures along squalene oxide in the reaction catalyzed by oxidosqualene coordinate of cyclization. It is interesting to note t cyclase (section 5.2). It is possible that conserved hydrogen and promiscuity are similariy balanced to accon bond interactions with the aspartic acid proton donor are multiple thioether cyclization reactions catalyzed important for function in squalene-hopene cyclase and cyclase, which unexpectedly adopts a double oxidosqualene synthase, and it is especially intriguing that comparable to that of a class I1 teipenoid cy~lase.'~~.~6 a polar tunnel with Glu-459 at its midpoint connects Asp- Class I and class 11 terpenoid cyclases and the cat& 455 to bulk solvent in oxidosqualene cyclase (Figure 41). antibody cyclases utilize the bulky aromatic residries ph In terms of its structural and chemical nature, this tunnel is ylalanine, tyrosine, and tryptophan to form their activ reminiscent of certain proton channels, and it could define a contours, and in each cyclase these residues also app trajectory for reprotonating the active site general acid at be oriented for the stabilization of carbocation inte~med' the end of the cyclization cascade.13' Given the ubiquitous Interestingly, structural studies with the catalytic anti occuirence of aspartic acid as a proton donor in the active cyclase 19A4 suggest that two aromatic residues are p sites of class 11 terpenoid cyclases, it is intriguing to speculate tioned to stabilize each site of developing positive or p that aspartic acid residues may participate in the protonadon positive charge as the polyene substrate undergoes cycliza of certain catalytic intermediates in the active sites of class (Figure 46),I4j and this also appears to be the case for- I terpe~loidcy~lases.~~.~~ stabilization of certain carbocation intermediates in In protonation-dependent class TI terpenoid cyclases such oxidosqualene cyclase mechanism (Figure 421."' As -sre as squalene-hopene cyclase and oxidosqualene cyclase,15" ously discussed in section 1, aromatic residues can p - crystal structures of enzyme-ligand complexes suggest that electrostatic stabilization of highly reactive cubo the active site contour appears to be preformed in the intermediates without danger of alkylation. It is notable t productive conformation required for the cyclization tem- both the naturally evolved terpene cyclases and the " plate. This also appears to be the case for the antibody naturally-evolved" antibody cyclases have converged to catalysts of polyene cyclization reactions discussed in section chemical strategy for carbocation stabilization. 6. In contrast, crystal structures of the ionization-dependent Finally, in view of the exquisite chemical colnpl class I tespenoid cyclases complexed with PP, or diphosphate- the cyclization cascade catalyzed by a terpenoid cy containing substrate analogues reveal that the productive is notable that given all the structural information active site contour is achieved only after the substrate is family of enzymes acquired in the past eight years, t bound. Compasison of Mg2+3-PP,-induced structural changes enzyme appears to plays surprisingly little chemical role in (f)-bornyl diphosphate synthase (section 2), trichodiene the cyclization cascade begins. While initial structure- synthase (section 3.11, and epi-aristolochene synthase (section hypotheses suggested roles for active site residues as g 3.2) reveal some common regions of structure that are bases or general acids, enzymological studies increas consistently involved in ligand-induced structural changes, suggest that the substrate itself can provide che? for example, the J-K loop, but there are more differences functionality required for specific general base/acid st than similarities. As noted by Whittington and colleagues,43 the cyclization cascade. For example, a carbon-c examples include the K-a1 loop, which participates in active double bond serves as a geileral base in the eaxa site closure mechanisms in (+)-bornyl diphosphate synthase synthase reaction (Figure 34),'24.126and the produce PPi a~ and trichodiene synthase, but not epl-aristolochene synthase, (or an associated water molecule) is considered to b and the N-terminus. which pasticipates in active site closure general base in cyclization reactions catalyzed by (+)-bo mechanisms in (+)-bornyl diphosphate synthase and epi- diphosphate ~ynthase,'~-~~trichodiene syntha~e,".~'and aristolochene synthase, but not trichodiene synthase. Diver- tolochene synthase8' and the chain elongation sea sity in substrate recognition and active site closure mecha- catalyzed by farnesyl diphosphate synthasc2' Thus, nisms may correlate with terpenoid cyclase product diversity, penoid cyclase itself may do little in the terpenoid cyc but additional structural data must be acquired with other reaction after initial carbocation formation terpenoid cyclases to assess this possibility. provide a productive template for tl-ie cyclization ca Regardless of whether the productive active site contour, and to stabilize reactive carbocation iniermedi the template for the isoprenoid cyclization reaction, is that these enzymes catalyze the most complex r induced by substrate binding, as evident for class I cyclases nature may be a consequence of the fact that these e such as (+I-bornyl diphosphate synthase and trichodiene do not extensively pasticipate in the cyclization che! synthase, or whether it is mostly preformed, as evident for Future structural and enzymological studies of teq

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