structure and function of cis-prenyl chain elongating enzymes

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precursors of steroid hormones in animals and plants. In addi-tion, three phytohormones, abscisic acid, gibberellins, andcytokinins, are also included in isoprenoids. Prenyl quininesare employed as electron carriers required for the mitochondr-ial respiratory chain and as cofactors in electron transportchains in plastids of higher plants. Protein prenylation, a

Structure and Function of cis-PrenylChain Elongating Enzymes

SEIJI TAKAHASHI, TANETOSHI KOYAMAInstitute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira,Aoba-ku, Sendai 980-8577, Japan

Received 30 March 2006; Revised 9 June 2006; Accepted 29 May 2006

ABSTRACT: All carbon skeletons of isoprenoids, whose chain lengths vary widely from geranyldiphosphate (C10) to natural rubber (C>10,000), are synthesized by sequential condensation of isopen-tenyl diphosphate with an allylic diphosphate through catalytic functions of a group of enzymes com-monly called “prenyltransferases.” Prenyltransferases are classified into two major groups, trans- or(E )-prenyltransferases and cis- or (Z )-prenyltransferases, according to the geometry of the prenylchain units in the products. From the year 1987, many genes encoding trans-prenyltransferases werecloned and clearly characterized. In contrast, the structure and detailed mechanism of cis-prenyl-transferase was completely unknown until the identification of a gene encoding the undecaprenyldiphosphate (UPP) synthase from Micrococcus luteus B-P 26 in 1998. Not only the primary but alsothe tertiary structure of the UPP synthase is quite different from that of the trans-prenyltransferases.Multiple alignment of the primary structures of cis-prenyltransferases identified from various organ-isms reveals five highly conserved regions. Site-directed mutagenesis of the conserved amino acidresidues in UPP synthases based on the crystal structure has elucidated the basic catalytic mecha-nisms. Moreover, comparison of the structures of short-, medium-, and long-chain cis-prenyltrans-ferases reveals important amino acid residues for product chain length determination, which enabledus to understand the regulation mechanism of the ultimate chain length among cis-prenyltransferases.© 2006 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 6: 194–205;2006: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20083

Key words: enzymes; protein structures; structure–activity relationships; terpenoids

The Chemical Record, Vol. 6, 194–205 (2006)

� Correspondence to: Tanetoshi Koyama; e-mail: [email protected]

Introduction

Isoprenoids, which are natural compounds derived biosyn-thetically from a five-carbon building unit, isopentenyl diphos-phate (IPP), are the most structurally diverse and abundantnatural products known, with more than 23,000 primary andsecondary metabolites. A variety of isoprenoids were identifiedas playing indispensable roles in all organisms.1–3 Sterols haveimportant functions not only as structural components of bio-logical membranes, controlling membrane fluidity, but also as

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post-translational modification mediated by covalent attach-ment of a farnesyl (C15) or geranylgeranyl (C20) moiety ontospecific proteins, such as Ras, trimeric G-proteins, and yeast-mating pheromone a-factor, is required for membrane associ-ation of these regulatory proteins that function in signaltransduction cascades of higher organisms. Dolichol plays animportant role as a glycosyl carrier lipid in the biosynthesis ofN-linked glycoproteins, O-linked oligosaccharides on yeastglycoproteins, C-linked mannose to tryptophan, and glyco-sylphosphatidylinositol-anchored protein.

From a metabolic viewpoint, the biosynthetic pathways ofisoprenoids can be divided into three phases: (i) formation ofIPP, (ii) condensation of IPP to synthesize linear isoprenoids,and (iii) condensation, cyclization, and modification of linearisoprenoids (Fig. 1). The first phase of the isoprenoid biosyn-thetic pathway is formation of the basic building unit of iso-prenoid IPP, which includes formation of dimethylallyldiphosphate (DMAPP) or isomerization of IPP into DMAPP.Up until 13 years ago, the acetate/mevalonate pathway (MVA

pathway), in which three molecules of acetyl-CoA condensesuccessively to form 3-hydroxyl-3-methylglutaryl-CoA(HMG-CoA) and followed to a key intermediate molecule,MVA, had commonly been considered as the sole metabolicpathway for the formation of IPP and DMAPP in all organ-isms. However, several lines of evidence indicated that themetabolic pathway of isoprenoids in certain eubacteria, plants,and green algae could not be solely explained by the MVApathway.4 Rohmer et al.5,6 discovered another metabolicpathway of IPP from eubacteria, which initiates with a con-densation of pyruvate and -glyceraldehyde 3-phosphate toform 1-deoxy--xylulose 5-phosphate (DXP). Because DXP is known to be used in other metabolic pathways, such as thebiosynthesis of pyridoxal phosphate and thiamin diphos-phate7–9, methyl--erythritol 4-phosphate (MEP), which isconverted from DXP by DXP reductoisomerase, is consideredto be the first committed precursor of the MVA-independentpathway.10 Therefore, this pathway is commonly termed as thenonmevalonate pathway or the MEP pathway. In the past

� Seiji Takahashi was born in 1972 in Fukushima, Japan. He obtained his Bachelor’s degreein 1995 and his Master’s degree in 1997 from Tohoku University, and his D.Sc. in 2001 fromTsukuba University under the direction of Professor Kazuo Shinozaki. After postdoctoral workat RIKEN with Professor Shinozaki for six months, he joined the Institute of MultidisciplinaryResearch for Advanced Materials, Tohoku University. He worked with Professor TanetoshiKoyama for four years as a Research Associate. In 2005, he moved to the Graduate School ofEngineering, Tohoku University, as a Research Associate. His current research interests focus onthe molecular mechanisms and physiological functions of prenyl chain elongating enzymes inhigher plants. �

� Tanetoshi Koyama was born in 1945 in Shizuoka, Japan. He graduated from Tohoku Uni-versity in 1968, where he also obtained his doctorate in 1973. After postdoctoral work at Stan-ford University with E. E. van Tamelen, he joined the Chemical Research Institute ofNon-Aqueous Solutions, Tohoku University, as a research associate in 1974. He was promotedto Associate Professor in the Department of Biochemistry and Engineering, Tohoku University,in 1994 and then to Professor in 1997 at the Institute for Chemical Reaction Science (renamedthe Institute of Multidisciplinary Research for Advanced Materials in 2000), Tohoku Univer-sity. He has been involved in enzymatic studies of isoprenoid biosynthesis, especially in the mole-cular analysis of enzymes related to isoprenoid biosynthesis. �

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decade, enzymes catalyzing in the MEP pathway were identi-fied from various organisms, and an overall metabolic pathwaywas elucidated.11,12

In the biosynthesis of isoprenoids, all carbon skeletons aresynthesized from linear isoprenoids at the second phase in iso-prenoid biosynthetic pathways. The carbon chain length ofnaturally occurring linear isoprenoids is widely distributedfrom geranyl diphosphate (GPP, C10) to natural rubber(C>10,000). Every linear isoprenoid is formed by sequential con-densation of IPP and allylic diphosphates with actions ofprenyl chain elongating enzymes, commonly called prenyl-transferases. In a broad sense, prenyltransferases include allenzymes that catalyze the transfer of allylic prenyl groups toacceptor molecules, such as IPP, aromatic intermediates ofquinones, or specific proteins. In this review, for convenience,prenyltransferase refers to prenyl diphosphate synthase thatcatalyzes the sequential condensation of IPP to allylic diphos-phates. The reactions catalyzed by prenyltransferases start bythe formation of allylic cations after the elimination ofpyrophosphate ions to form allylic prenyl diphosphate, fol-lowed by addition of an IPP with stereospecific removal of aproton at the 2-position. With respect to the geometry of theprenyl chain units in the products, prenyltransferases can beclassified into two major groups, i.e., trans- or (E )- and cis- or(Z )-prenyltransferases.12 The biosynthesis of linear isoprenoidscan be divided into two phases (Fig. 1). In the first phase,short-chain allylic diphosphates, GPP (C10), farnesyl diphos-

phate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20)are formed by the action of trans-prenyltransferases. These all-trans-short chain prenyl diphosphates are then employed asallylic primer substrates for additional IPP condensation withtrans- or cis-configuration by trans- or cis-prenyltransferases,respectively. The prenyltransferases responsible for each linearisoprenoid with a specific number of isoprene units strictly rec-ognize the prenyl chain lengths of the allylic substrates andultimate products. From the standpoints of enzymology andorganic chemistry, this property of prenyltransferases is one ofthe most interesting research topics regarding the catalyticmechanisms of prenyl chain elongating enzymes. The absolutestereochemistry of reactions catalyzed by each type of prenyl-transferase with respect to the face of the double bond of IPPduring the carbon–carbon bond formation was revealed using(all-E)-heptaprenyl diphosphate synthase (trans-prenyltrans-ferase) and UPP synthase (cis-prenyltransferase) from Bacillussubtilis.13,14 The only difference between the reaction catalyzedby trans- and cis-prenyltransferases is the prochirality of theproton that is eliminated from the 2-position of IPP, i.e., pro-R for trans-prenyltransferase and pro-S for cis-prenyltrans-ferase (Fig. 2), suggesting a similar structural property in thecatalytic centers of cis- and trans-prenyltransferases. However,recent molecular analyses and crystal structure determinationsof M. luteus B-P 26 UPP (C55) synthase, which catalyzessequential cis-condensation of IPP, showed that not only the primary but also the three-dimensional (3D) structure of

Fig. 1. Biosynthetic pathways of linear isoprenoids catalyzed by trans- and cis-prenyltransferases.MEP, methyl-D-erythritol 4-phosphate; MVA, acetate/mevalonate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP,methyl-D-erythritol 4-phosphate; HMG, 3-hydroxyl-3-methylglutaryl; OPP, PP, corresponding diphosphate ester−OP2O6

3−.



cis-prenyltransferases was totally different from that of trans-prenyltransferases.15–17

In this review, we outline the recent progress of molecularanalysis for structure, catalytic mechanism, and product chainlength regulation of cis-prenyltransferases.

Structure of cis-Prenyltransferases

After the first cDNA cloning of rat liver FPP synthase byClarke et al.,18 many kinds of genes responsible for trans-prenyltransferases were cloned and characterized from variousorganisms.1,2,19 The accumulation of information for theprimary structures of trans-prenyltransferase revealed sevenhighly conserved regions in the trans-prenyltransferases amongspecies.12 Site-directed mutational analysis of amino acids inthe conserved regions revealed the involvement of the con-served residues in catalytic function and substrate recognitionof trans-prenyltransferases (Fig. 3). In particular, two aspartate-rich motifs, DDX2–4D and DDXXD in regions II and VI,respectively, are typical for trans-prenyltransferases and essen-tial for catalytic function as well as substrate binding.2,19 In1994, Tarshis et al.20 reported the crystal structure of avian FPPsynthase as the first determination of prenyltransferase (Fig. 2).Avian FPP synthase is a homodimer protein, and each

monomer subunit is composed of thirteen α-helices. Amongthem, 10 α-helices compose a large central cavity for a catalyticcenter, in which two aspartate-rich motifs are located on oppo-site sidewalls of the cavity. Tarshis et al.21 also determined thecrystal structure of the avian FPP synthase binding allylic sub-strate FPP, showing that the diphosphate group of FPP isbound to the first aspartate-rich motif (FARM) in region IIthrough bridges of two magnesium ions. More recently, thestructure of the ternary complex of FPP synthase fromEscherichia coli containing IPP and a noncleavable DMAPPanalogue was determined. The structure revealed a dynamicconformational change of active site at the C-terminus andloops between α4–α5 and α9–α10 that contain conservedresidues of trans-prenyltransferases.22 These structural analysesprovided much information about the catalytic mechanism oftrans-prenyltransferases at the molecular level.

In contrast, we had never been successful in cloning geneencoding cis-prenyltransferases with the strategy of isolatinghomologous genes of trans-prenyltransferases different fromthe stereochemical similarity of the reactions catalyzed by eachtype of prenyltransferase. In 1998, Shimizu et al.15 applied aconvenient method, the so-called “colony autoradiographymethod,” which was developed by Raetz et al.,23 to screencolonies of E. coli overexpressing cis-prenyltransferase. In thismethod, E. coli colonies harboring a prenyltransferase gene

Fig. 2. Schematic diagram of the reactions catalyzed by trans-prenyltransferase and cis-prenyltransferase and com-parison of tertiary structures of farnesyl diphosphate (FPP) synthase from avian liver20 and undecaprenyl diphosphate(UPP) synthase from Micrococcus luteus B-P 2617.IPP, isopentenyl diphosphate; OPP, PP, corresponding diphosphate ester −OP2O6

3−.



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from M. luteus B-P 26 were functionally screened as cells thatshow high incorporation of a radiolabeled substrate, IPP,resulting in the first identification of the cis-prenyltransferasegene encoding UPP synthase of M. luteus B-P 26. UPP syn-thase catalyzes the sequential cis-condensation of eight IPPswith FPP to biosynthesize UPP, which is a precursor of unde-caprenyl phosphate, an indispensable glycosyl carrier lipid inbacterial cell wall biosynthesis.24 Subsequently, Apfel et al.16

also succeeded in isolating the gene for UPP synthase from E.coli, Haemophilus influenzae, and Streptococcus pneumonia.After the first report of gene cloning of UPP synthase, manygenes among the unknown ones submitted in databases wereidentified as cis-prenyltransferases that have similarities withthe UPP synthase. In particular, one of the rer mutants of Sac-charomyces cerevisiae,25 which has a defect in endoplasmic retic-ulum (ER) protein sorting, rer2, was shown to have a mutationin the gene that shows high homology with UPP synthase.26

RER2 encodes a dehydrodolichyl diphosphate (DedolPP) syn-thase that is responsible for the biosynthesis of the precursorof dolichol, which plays indispensable roles as a sugar carrierin protein glycosylation in ER. To date, homologous genes forUPP synthase have been isolated from various species, such asmycobacteria,27 archae,28 higher plants,29–31 and human.32

It is surprising that the primary structures of cis-prenyl-transferases are quite different from those of trans-prenyltrans-ferases. Multiple alignment of deduced amino acid sequencesreveals five highly conserved regions among species2 (Figs. 3and 4A). In these conserved regions of cis-prenyltransferase, nocorresponding amino acid residues with those of trans-prenyl-transferase were found. In particular, Asp-rich motifs, whichare typically included not only in trans-prenyltransferases butalso in other isoprenoid biosynthetic enzymes, such as IPP iso-

merases and terpene synthases, are not found in cis-prenyl-transferases, suggesting that cis-prenyltransferases may be anevolutionarily distinct protein family from other isoprenoidbiosynthetic enzymes involving trans-prenyltransferases.

To gain further insight into the molecular mechanism ofcis-prenyltransferase, Fujihashi et al.17 determined the crystalstructure of UPP synthase from M. luteus B-P 26 at 2.2Å resolution, which was the first report about a 3D structure forcis-prenyltransferases. This enzyme acts as a homodimer com-posed of a set of 29-kDa subunits. Each monomer contains sixparallel β-strands forming a central β-sheet core, which is sur-rounded by five of the seven α-helices. Surprisingly, the crystalstructure is also completely different from that of trans-prenyl-transferase, FPP synthase (Fig. 2). Subsequent to the determi-nation of the crystal structure of UPP synthase from M. luteusB-P 26, the 3D structure of E. coli UPP synthase wasrevealed,33 showing a nearly similar structure to that for M.luteus B-P 26 UPP synthase. Furthermore, the structure of theUPP synthase is quite different from that of a common struc-tural motif called “isoprenoid synthase fold,”34 which was sug-gested for inclusion in most of the enzymes related toisoprenoid biosynthesis. The isoprenoid synthase fold contains10–12 mostly antiparallel α-helices, which can be seen in thestructures of the FPP synthase,20 sesquiterpene cyclase, squa-lene synthase, and protein farnesyl transferase.35–37

A large hydrophobic cleft on the molecular surface isfound in the crystal structure of the UPP synthase subunit.The cleft is composed of two α-helices, helix-2 and helix-3,and two β-sheets, sheet-2 and sheet-4, in which most of theconserved amino acid residues in the five conserved regions ofcis-prenyltransferase are located; the interior of the cleft con-sists mostly of hydrophobic residues. Therefore, this cleft is

Fig. 3. Comparison of conserved regions between trans- and cis-prenyltransferases. Proposed functions of amino acidresidues in the conserved regions are indicated.IPP, isopentenyl diphosphate.



considered to function in recognition of the allylic substrate,FPP, which has a large hydrophobic prenyl chain, and accom-modation of the elongated hydrophobic prenyl intermediates.At the entrance of the cleft, which corresponds to conservedregion I, a typical motif for the phosphate group recognitioncalled “structural P-loop”38 is found. In the crystal structure, asulfate ion, which was used as a precipitant in the crystalliza-tion, is bound to this motif, suggesting that the negativelycharged diphosphate moiety of the allylic substrate FPP is rec-ognized by this structural P-loop motif. Indeed, basic aminoacid residues including highly conserved amino acid residues(Arg-197 and Arg-203) form a positively charged cluster nearthe structural P-loop motif, which seems to be suitable for thebinding of the phosphate group of another substrate IPP. Inthe crystal structure of M. luteus B-P 26 UPP synthase withoutsubstrates, residues from Ser-74 to Val-85 downstream ofsheet-2 in conserved region III could not be defined becauseof high flexibility,17 suggesting that the flexible domain isimportant for the binding of substrates and catalytic function.

Recently, the crystal structures of UPP synthase from E.coli with allylic or homoallylic substrates were determined.39–40

These structural analyses also provided insight into the rela-tionship between the structure and catalytic function of cis-prenyltransferase. Thus, structural properties are discussed inthe following section along with the molecular mechanisms ofcatalytic function.

Catalytic Mechanism of cis-Prenyltransferases

In contrast to trans-prenyltransferases, which were cloned adecade before cis-prenyltransferases, elucidation of the molec-ular mechanism of cis-prenyltransferases has not yet been com-pleted. In an earlier study, a random mutagenetic study of M.luteus B-P 26 UPP synthase41 was performed to discover thecritical amino acid residues in the catalytic activity, revealingthe importance of Asn-77 and Trp-78 in region III, which werehighly conserved residues among the cis-prenyltransferases.Substitutions of Asn-77 for Ala, Asp, or Gln resulted in 102-to 103-fold smaller kcat values than that of wild type withoutsignificant change in Km values for IPP and allylic substrates,indicating the significance of Asn-77 in catalytic activity. Incontrast, replacement of Trp-78 with charged amino acidresidues leads to a 2- to 8-fold lower kcat value than that of wildtype and a remarkable decrease in Km for FPP. These resultssuggest that the aromatic residues of Trp-78 form a hydropho-bic interaction with the prenyl chain of FPP. Pan et al.42

focused on the conserved acidic residues among cis-prenyl-transferases because no typical aspartate-rich motif DDXXDis found in cis-prenyltransferases. Comprehensive site-directedmutagenesis of the conserved Asp and Glu on E. coli UPP syn-thase revealed the significance of Asp-26 in region I for cat-

alytic activity, which corresponds to Asp-29 of M. luteus UPPsynthase. Furthermore, both Asp-150 in region IV and Glu-213 in region V were shown to be important for IPP binding.However, these conserved acidic residues in cis-prenyltrans-ferases are not too critical for substrate binding and catalyticactivity as are the DDXXD motifs in trans-prenyltransferases,suggesting a different mechanism for substrate binding in cis-prenyltransferases.

A detailed model for substrate binding and a catalyticmechanism of cis-prenyltransferase were brought about by site-directed mutagenesis according to the crystal structure ofcis-prenyltransferases. Kharel et al.43 performed site-directedmutagenesis of the highly conserved charged residues in regionV, which form a high electrostatic potential region, in thecrystal structure of M. luteus UPP synthase (Fig. 4). In addi-tion, the Phe–Ser motif in region III, which is also highly con-served, was substituted with Ala because the Phe–Ser motif islocated at the position adjacent to the high-electrostatic-poten-tial region in region V. Substitution of Arg-197 and Arg-203with Ser and Glu-216 with Gln in conserved region V caused7- to 11-fold increases of Km for IPP and 18- to 2000-folddecreases of kcat, indicating the critical roles of these chargedresidues in catalytic function as well as in IPP binding. Accord-ing to these results and the crystal structure of UPP synthase,a hypothetical binding model for IPP to the UPP synthase waspostulated.43 Diphosphate moiety of IPP may be recognizeddirectly with the positively charged residues of Arg-197 andArg-203 and indirectly with the carbonyl group of Glu-216,which extends from another monomer, through an Mg2+

bridge. Phe-73 in the Phe–Ser motif may function in theholding of the hydrocarbon moiety of homoallylic substrate

Fig. 4. (A) Multiple alignment of amino acid sequences of seven cis-prenyl-transferases: Rv1086: Z,E-FPP synthase from Mycobacterium tuberculosis(Accession No.: D70895), Rv2361c: DecPP synthase from M. tuberculosis(H70585), Micrococcus luteus: undecaprenyl diphosphate (UPP) synthase fromM. luteus B-P 26 (BAA31993), Escherichia coli: UPP synthase from E. coli(Q47675), Rer2p: dehydrodolichyl diphosphate synthase from Saccharomycescerevisiae (BAA36577), Srt1p: dehydrodolichyl diphosphate synthase from S.cerevisiae (NP_013819), HDS: dehydrodolichyl diphosphate synthase fromhuman (BAC57588). Upper lines indicate conserved regions. Residues withmore than 70% identity are boxed, and all residues are colored as follows: non-polar (G, A, V, L, I, P, F, M, W, C), yellow; uncharged polar (N, Q, S, T, Y),green; acidic (D, E), red; basic (K, R, H), blue. Blue and purple underlinesindicate the corresponding positions of β-sheets and α-helices as revealed fromthe crystal structure of UPP synthase from M. luteus B-P 26,17 respectively.Red triangles and blue triangles indicate residues that are characteristic forshort- and long-chain cis-prenyltransferase, respectively. (B) Classification ofcis-prenyltransferases with respect to product chain lengths. These enzymescatalyze cis-condensation of IPP (isoprene unit, C5) onto an allylic diphos-phate, all-E-prenyl diphosphate. Grey lines indicate numbers of isoprene unitsof representative allylic substrates for each cis-prenyltransferase. Red arrowsindicate numbers of isoprene units of representative ultimate products, whichare condensed with cis-configuration by each enzyme.

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IPP, while Ser-74 may bind to the diphosphate group of IPPwith hydrogen bonding. A critical function of the structuralP-loop motif as well as Asp-29 and Arg-33 in the binding of pyrophosphate group of allylic substrate FPP, which is proposed by the crystal structure of M. luteus B-P 26 UPP synthase, was also confirmed by studies of site-directed mutagenesis.41–44

Recently, Chang et al.39 succeeded in determining thecomplex crystal structure of E. coli UPP synthase and the allylicsubstrate FPP. More recently, Guo et al.40 determined thecrystal structure of E. coli UPP synthase in complex with Mg2+,IPP, and farnesyl thiopyrophosphate, a low reactive analogueof FPP in which the bridging oxygen atom between farnesyland pyrophosphate group is replaced with a sulfur atom. Thesecomplex structures clarified the models for the catalytic mech-anism of UPP synthase, which were proposed by the studiesof site-directed mutagenesis (Fig. 5). Comparison of the crystalstructures of UPP synthase with and without substratesrevealed the dynamic conformational change between the open(apoenzyme and product-bound) and closed (substrate-bound)forms. The flexible loop downstream of sheet-2, which couldnot be defined in the crystal structure without substratesbecause of high flexibility,17 becomes visible in the new struc-tures with both allylic and homoallylic substrates. Thepyrophosphate group of FPP is stabilized by the structural P-loop motif, and the hydrocarbon tail of FPP is bound by thehydrophobic residues in helix-2 and helix-3, which form thehydrophobic interior of the large hydrophobic cleft. Occupa-

Fig. 5. (A) Overall catalytic center of a structural model for Micrococcus luteusB-P 26 UPP synthase. The model was built based on the crystal structure ofE,E–farnesyl diphosphate (FPP) complex of undecaprenyl diphosphate (UPP)synthase from Escherichia coli (Protein Data Bank No.: 1V7U). Then, struc-tures of E,E-FPP and isopentenyl diphosphate (IPP), which were identifiedfrom the complex structure of IPP and the D26A mutant of E. coli UPP syn-thase (Protein Data Bank No.: 1X09), were superimposed on the structuralmodels. Amino acid residues mutated in the A72L/F73L/W78L/F223Hmutant are indicated in red and are overlapped with residues in the wild-typeUPP synthase. The structural P-loop motif, proposed to function in thebinding of allylic substrates such as E,E-FPP, and charged residues includingArg-197 and Arg-203 are indicated. (B) Large hydrophobic cleft of M. luteusB-P 26 UPP synthase with or without FPP. Models were built based on thecrystal structure of E,E-FPP complex of UPP synthase from E. coli (ProteinData Bank No.: 1V7U). Then, the structure of E,E-FPP was superimposedon the models. Side chains are colored as follows: nonpolar, gray; unchargedpolar, yellow; acidic, red; basic, blue. (Left) Large hydrophobic cleft of M.luteus B-P 26 UPP synthase (Protein Data Bank No.: 1F75) consisting of helix-2, helix-3, sheet-2, and sheet-4. Only side chains facing toward the inside ofthe hydrophobic cleft are indicated. (Right) Structural model of the largehydrophobic cleft of M. luteus B-P 26 UPP synthase with E,E-FPP. Only sidechains facing toward the inside of the hydrophobic cleft are indicated. Thewhite arrow indicates the predicted direction of the prenyl chain elongation.The circle indicates regions in which the extra amino acid residues, EKE orRAKDY, were introduced.

�

tion of the allylic substrate binding site leads to the confor-mational change of E. coli UPP synthase from the open to theclosed forms, in which helix-3 is kinked to be closer to theallylic substrate binding domain compared with the structurewithout substrates.33 Leu-85, Leu-88, and Phe-89 in kinkedhelix-3 participate in the FPP binding, which is considered tobe critical for regulation of UPP synthase activity. In the crystalstructure of E. coli UPP synthase with FPP and IPP, Mg2+ isoctahedrally coordinated by the pyrophosphate group of FPP,three water molecules, and the carboxylate residue of Asp-26,which corresponds to Asp-29 in M. luteus UPP synthase.However, in the crystal structure of the D26A mutant with



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substrates,40 Mg2+ is not in direct contact with the pyrophos-phate group of FPP but is associated with the binding site forthe pyrophosphate group of IPP. These results strongly suggestthe indispensable role of Asp-26 in the catalytic mechanism asan assistant of the Mg2+ migration from IPP to FPP, which mayinitiate the condensation reaction by ionization of thepyrophosphate group of FPP. According to the complex struc-ture, the HS atom at the 2-position of IPP, which is removedin the cis-condensation, faces outside of the catalytic center.The candidates for the basic residues function in the stere-ospecific removal of the proton are Asn-74 and Ser-71, whichcorrespond to Asn-77 and Ser-74 of UPP synthase from M.luteus B-P 26, respectively. The site-directed mutageneses ofthese residues, showing a substantial decrease of the kcat

values,33,42 indicate the significant role played by these residuesin the catalytic activity of UPP synthase. Taken together, thebinding mode of cis-prenyltransferase for the substrates, FPPand IPP, is totally different from that of trans-prenyltransferase,which employs the Asp-rich motif for substrate bindingthrough the Mg2+ bridge.

Chain Length Determination Mechanism for cis-Prenyltransferase

One of the most interesting research topics on prenyl chainelongating enzymes is to understand the mechanisms by whichindividual prenyltransferases recognize the prenyl chainlengths of allylic substrates and products. Using a series ofeffective random chemical mutagenesis, Ohnuma et al.19,45

showed that the ultimate product chain length of trans-prenyl-transferases was regulated by bulky residues located upstreamof the FARM, which is one of the most highly conservedregions among trans-prenyltransferases. The bulky residuesfunction as a floor of the catalytic pocket for the growing iso-prenoid chain to block further elongation. Based on crystalstructure and mutagenetic studies of the avian FPP synthase,Tarshis et al.21 concluded that allylic diphosphate boundthrough Mg2+ to Asp residues in the FARM motif. On the con-trary, mechanisms for determination of the ultimate productchain length of cis-prenyltransferases were supposed to be dif-ferent from that of trans-prenyltransferases because not onlythe primary structure but also the 3D structure is totally dif-ferent between cis- and trans-prenyltransferases. Althoughmutational analysis of highly conserved residues and determi-nation of crystal structures of UPP synthase have enabled usto understand the basic catalytic mechanisms of cis-prenyl-transferases,3,46 mechanisms for determination of the ultimateproduct chain length of cis-prenyltransferases have not beenelucidated.

Ko et al.33 reported that replacement of Leu-137 of E. coliUPP synthase, which is located at the “bottom” of the

hydrophobic cleft, with Ala resulted in one-unit elongation ofthe ultimate chain length of Z,E-mixed polyisoprenoids, pro-ducing C55 and C60 as major products in the presence of 0.1%Triton X-100. In addition, they indicated that the mutantL137A produced C70 and C75 as major products in a reactionfor 96h without Triton X-100. Based on these results, theyproposed that Leu-137 functioned as the floor of the tunnelto block further elongation of polyprenyl products. This pro-posed model seems to be analogous to the chain length deter-mination mechanism of trans-prenyltransferases, in whichbulky residues located upstream of FARM play an importantrole in determining the ultimate prenyl chain length, com-posing a suitable size for the pocket for growth of the iso-prenoid chain. According to the model, we constructed andanalyzed a mutant of M. luteus B-P 26 UPP synthase L140A,which corresponded to the E. coli UPP synthase mutantL137A, with the result that the mutant also produced C55 andC60 as major products in the presence of Triton X-100 (unpub-lished data). Elongation of the ultimate product to C70–75 inthe reaction of the mutant L137A without Triton X-100 seemsto depend on the reaction conditions in vitro. Matsuoka et al.47

reported that chain length distribution of products fromDedolPP synthase in microsomal fractions of rat liver could beaffected by reaction conditions in vitro such as detergents andphospholipids.

However, recent reports on cloning and characterizationof cis-prenyltransferases from various organisms showed thatthe specificities of product chain lengths primarily dependedon different enzymatic properties attributable to the structuraldiversity of each enzyme. With respect to product chain length,the cis-prenyltransferases identified so far were classified intothree subfamilies: (i) short-chain (C15), (ii) medium-chain(C50–55), and (iii) long-chain (C70–120) cis-prenyltransferases(Fig. 4B). Z,E-FPP (C15) synthase from Mycobacterium tuber-culosis, Rv1086, is the only enzyme identified as a short-chaincis-prenyltransferase, which catalyzes cis-condensation of oneIPP with GPP (C10).27 Medium-chain cis-prenyltransferases are represented by UPP synthase, which catalyzes cis-conden-sation of eight molecules of IPP with E,E-FPP (C15). Thisenzyme is responsible for the biogenesis of undecaprenyl phos-phate, an indispensable glycosyl carrier lipid in bacterial cellwall biosynthesis. Z,E-mixed decaprenyl diphosphate (Z,E-DecPP, C50) synthase from M. tuberculosis, Rv2361 is also cat-egorized in this subfamily.27 Most DedolPP synthases ineukaryotes catalyzing synthesis of the precursors of the sugarcarrier lipid dolichol during biosynthesis of glycoproteins arecategorized as long-chain cis-prenyltransferases. Srt1p andRer2p from S. cerevisiae26,48 and HDS from human32 areincluded in this subfamily. In order to investigate regionsimportant for determination of product chain length in cis-prenyltransferases, Kharel et al.49 explored the characteristicresidues of short- or long-chain cis-prenyltransferase subfami-


203

lies based on comparisons between the primary structures ofcis-prenyltransferases identified so far and the crystal structuresof UPP synthases. They introduced mutations in regions of M.luteus B-P 26 UPP synthase, which correspond to the charac-teristic residues (Fig. 4A).

Rv1086 from M. tuberculosis, which is the only enzymecloned and identified as a short-chain cis-prenyltransferase, hasLeu residues at positions 84, 85, and 90 instead of the corre-sponding Ala, Phe, and Leu/Trp in the conserved Region IIIof other cis-prenyltransferases. The corresponding residues inM. luteus B-P 26 UPP synthase, i.e., Ala-72, Phe-73, and Trp-78, in the highly conserved region III are located at the edgeof the large hydrophobic cleft. When E,E-FPP and GPP wereused as allylic substrates, the M. luteus UPP synthase mutants,A72L/F73L (LL) and A72L/F73L/W78L (LLL), resulted inshorter (C25–40 as major products) and even shorter (C20–35 asmajor products) polyisoprenoids, respectively, than did wild-type UPP synthase, which produces C55–60 polyisoprenoids. Inthe crystal structure of E. coli UPP synthase bound with theallylic substrate E,E-FPP39,40, Ala-69, which corresponds toAla-72 of M. luteus B-P 26 UPP synthase, is located close tothe ω-end carbon, C-14 of E,E-FPP at a distance of 3.2Å. Thissuggests that the bulky alkyl group of Leu-72 in the mutantsor Leu-84 in Rv1086 might interfere with cis-addition of IPPonto E,E-FPP or Z,E-FPP (Fig. 5A). In contrast, Phe-73 andTrp-78 of M. luteus B-P 26 UPP synthase, corresponding toLeu-85 and Leu-90 of Rv1086, respectively, are not close tothe binding site for E,E-FPP but are placed on a flexibleloop.17,39,40 Substitutions of the highly conserved Phe-73 andSer-74 of M. luteus B-P 26 UPP synthase into Ala result in 32-and 16-fold increases in the Km value for IPP, and 16- and 12-fold decreases in the kcat value, respectively,43 indicating thatthe flexible domain in the conserved region III is importantboth for the binding of IPP in the proper direction and forcatalytic function. The double mutant A72L/F73L also showsan 88-fold higher Km value for IPP compared with the wild-type enzyme. However, the triple mutant A72L/F73L/W78Lshows a sixfold increase in the Km value for IPP. These resultssuggest that replacement of Trp-78 for Leu was necessary forthe creation of a proper IPP binding domain when Ala-72 andPhe-73 were replaced with Leu residues, which correspondedto Leu-84, Leu-85, and Leu-90, respectively, in Rv1086.

Furthermore, His-237 in Rv1086 downstream of the con-served Region V was found at the corresponding position forthe Leu/Phe residue in other cis-prenyltransferases. Substitu-tion of Phe-223 of M. luteus B-P 26 UPP synthase, which cor-responds to His-237 in Rv1086, with His dramaticallydecreased the catalytic activity of UPP synthase when E,E-FPPwas used as allylic substrate. However, the quadruple muta-tions A72L/F73L/W78L/F223H (LLLH) can accept GPP asan allylic substrate, producing shorter prenyl products, C45–55

and C15. According to the crystal structure of UPP synthase,

His-237 of Rv1086 is considered located near the structural P-loop motif, which recognizes the diphosphate group of allylicsubstrates,17,44 suggesting that His-237 in Rv1086 might affectthe binding affinity for the allylic substrate with the structuralP-loop motif.

Multiple alignment of cis-prenyltransferases also revealedthat long-chain cis-prenyltransferases, such as Srt1p and Rer2pfrom S. cerevisiae26,48 and HDS from human32, have three toseven extra amino acid residues downstream of the conservedregion III (Fig. 4A). This position corresponds to helix-3 ofthe M. luteus B-P 26 UPP synthase. Helix-3 participates in thecreation of the hydrophobic cleft, which is considered toaccommodate the elongated prenyl intermediates.17 Themutant of M. luteus B-P 26 UPP synthase, EKE, which con-tains insertion corresponding exactly to the extra amino acidresidues of DedolPP synthases from human (HDS, positions107 to 109), produce relatively longer prenyl products withcarbon chain lengths of C55–70. Moreover, the mutant RAKDY,which contains insertion corresponding exactly to the extraamino acid residues of DedolPP synthases from yeast (Srt1p,positions 148 to 152), gives even longer prenyl products withchain lengths of C60–75. Insertion of five Ala residues instead ofthe peptide RAKDY does not cause a significant change in thelength of prenyl products, suggesting the significance of somecharged or polar residues at the proper positions rather thanexpansion of the interior space of the hydrophobic cleft. Asmentioned earlier, in the crystal structure of E. coli UPP syn-thase bound with E,E-FPP, helix-3 is kinked to be closer to theE,E-FPP binding domain compared with the structure withoutsubstrates.39,40 The interior of the large hydrophobic cleft, sur-rounded by sheet-2, sheet-4, helix-2, and helix-3, consistsmostly of hydrophobic residues. Hydrophobic residues onkinked helix-3 in the closed conformation may function as aguide rail to introduce the elongating hydrophobic prenylchain in the proper direction (Fig. 5B). Moreover, the domainin which we introduced extra amino acid residues corre-sponded to the hinge region of the kinked helix-3. Chang etal. reported the crystal structure of UPP synthase from E. coliin complex with Triton X-100. Interestingly, in this structure,two molecules of Triton X-100 are bound in the catalyticcenter and the large hydrophobic cleft, folding with a suitabletorsion angle for accommodation in the hydrophobic cleft.They supposed that the conformation of Triton X-100 mole-cules, which interact with hydrophobic residues in the cleft,may mimic that of the hydrophobic moiety of UPP, and pro-posed the conformation of UPP folded in the cleft. In thestructure, the polyprenyl chain is also folded back at the posi-tion corresponding to the hinge region of the kinked helix-3.Therefore, it is postulated that charged residues inserted at thehinge region of helix-3 might control the bending direction ofthe growing hydrophobic prenyl chain along the hydrophobicinterior of helix-3 so that the hydrophobic cleft could accom-




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modate the bulk of the prenyl chain to fit a suitable size duringenzymatic elongation. The fact that residues localized on helix-3 show a wide diversity while most residues constituting thehydrophobic cleft are highly conserved among cis-prenyltrans-ferases also supports the hypothesis.

Conclusion and Perspectives

With the molecular cloning of UPP synthase from M. luteusB-P 26 in 1998 as a turning point, the study of prenyl chainelongation enzymes turned in a new direction. Detailed analy-ses of the relationships between protein structure and catalyticmechanism of a new family of isoprenoid biosyntheticenzymes, cis-prenyltransferase, and comparative studies of bothtypes of prenyl chain elongation enzymes are of great interestas aspects of molecular evolution. In addition, the chain lengthdetermination mechanism of cis-prenyltransferase is quiteimportant because biological materials composed by thepolymer of IPP with cis-configuration, such as natural rubber,can be employed for the development of novel functionalmaterials. Recently, our group reported the molecular cloningof two cDNAs, HRT1 and HRT2, encoding cis-prenyltrans-ferase from the latex of Hevea brasiliensis, which is almost thesole source for commercial natural rubber production.31 Thepredominant expression of these genes in latex suggests a spe-cialized function of HRTs in latex, i.e., cis-1,4-polymerizationfor the carbon backbone of natural rubber. In vitro prenyl-transferase assay using recombinant HRT protein revealed thecatalytic activity for the production of polyisoprenoids with acarbon length of ∼C100. However, in the presence of a latexfraction, HRT shows high prenyltransferase activity, produc-ing polyisoprenoids with higher molecular size, which corre-sponds to natural rubber. These results suggest the existence ofmechanism for functional conversion in the chain lengthdetermination of cis-prenyltransferase. Further investigationsmight provide a general rule for the regulation of the carbonchain length of products from cis-prenyltransferase, and mayenable us to manipulate the chain length determination mech-anism of the enzyme so as to synthesize polyisoprenoids withvarious chain lengths.

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