Báo cáo khoa học: The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction pptx

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The product chain length determination mechanism oftype II geranylgeranyl diphosphate synthase requiressubunit interactionMotoyoshi Noike1,2, Takashi Katagiri1, Toru Nakayama1, Tanetoshi Koyama2, Tokuzo Nishino1andHisashi Hemmi11 Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Miyagi, Japan2 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi, Japan(All-E) prenyl diphosphate synthase catalyzes the con-secutive condensation of isopentenyl diphosphates(IPP) with allylic prenyl diphosphates to yield the finalproduct with a specific prenyl chain length [1,2]. Thechain length of the product must be tightly controlledbecause polymerization of isoprene units is the keyreaction responsible for the tremendous variety ofnaturally occurring isoprenoid compounds (> 50 000)[3]. For example, many important compounds, such ascarotenoids, tocopherols, diterpenes, chrolophyll andarchaeal membrane lipids, are synthesized from gera-nylgeranyl diphosphate (GGPP; C20). On the otherKeywordsfarnesyl diphosphate synthase;geranylgeranyl diphosphate synthase;isoprenoid; mutagenesis; prenyltransferaseCorrespondenceH. Hemmi, Department of AppliedMolecular Bioscience, Graduate School ofBioagricultural Sciences, Nagoya University,Furo-cho, Chikusa-ku, Nagoya, Aichi464-8601, JapanFax: +81 52 789 4120Tel: +81 52 789 4134E-mail: hhemmi@agr.nagoya-u.ac.jp(Received 21 February 2008, revised 2 June2008, accepted 4 June 2008)doi:10.1111/j.1742-4658.2008.06538.xThe product chain length determination mechanism of type II geranyl-geranyl diphosphate synthase from the bacterium, Pantoea ananatis, wasstudied. In most types of short-chain (all-E) prenyl diphosphate synthases,bulky amino acids at the fourth and/or fifth positions upstream from thefirst aspartate-rich motif play a primary role in the product determinationmechanism. However, type II geranylgeranyl diphosphate synthase lackssuch bulky amino acids at these positions. The second position upstreamfrom the G(Q/E) motif has recently been shown to participate in themechaism of chain length determination in type III geranylgeranyl diphos-phate synthase. Amino acid substitutions adjacent to the residues upstreamfrom the first aspartate-rich motif and from the G(Q/E) motif did notaffect the chain length of the final product. Two amino acid insertion inthe first aspartate-rich motif, which is typically found in bacterial enzymes,is thought to be involved in the product determination mechanism. How-ever, deletion mutation of the insertion had no effect on product chainlength. Thus, based on the structures of homologous enzymes, a new lineof mutants was constructed in which bulky amino acids in the a-helixlocated at the expected subunit interface were replaced with alanine. Twomutants gave products with longer chain lengths, suggesting that type IIgeranylgeranyl diphosphate synthase utilizes an unexpected mechanism ofchain length determination, which requires subunit interaction in thehomooligomeric enzyme. This possibility is strongly supported by therecently determined crystal structure of plant type II geranylgeranyldiphosphate synthase.AbbreviationsDMAPP, dimethylallyl diphosphate; FARM, first aspartate-rich motif; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; GGPP,geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; GPS, geranyl diphosphate synthase;IPP, isopentenyl diphosphate; SARM, second aspartate-rich motif.FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3921hand, farnesyl diphosphate (FPP; C15) is the precursorof steroids, sesquiterpenes and heme a. Moreover, FPPis the usual allyic primer substrate for prenyl elonga-tion reactions, which yield longer-chain prenyl diphos-phates as the precursors of respiratory quinones,dolichol and natural rubber, although some organismsalso use GGPP for the same purpose. Longer-chain(all-E) prenyl diphosphates (up to C60) are utilized forthe biosynthesis of various respiratory quinones, whichhave been used to classify the microorganisms. GGPPand FPP are also utilized for protein modification,although they modify very different classes of acceptorproteins. Rab family G proteins are geranylgeranyl-ated, whereas farnesylation typically occurs on Rasproteins. In addition, geranyl diphosphate (GPP; C10)is the precursor of volatile monoterpenes and also isused to modify secondary metabolites.The mechanism of prenyl-chain elongation, andtherefore of product determination in (all-E) prenyldiphosphate synthases, which share many conservedsequences in spite of their different reaction products,has been investigated previously. The enzymes are con-structed mainly of a-helices, which form a large reac-tion cavity per a subunit [4–12]. Most of the enzymesare homodimeric proteins, although some enzymesconsist of heterodimers with little homology betweenthe subunits [1]. A few mammalian enzymes are knownto form oligomers [13,14]. The highly conserved motifsof the enzymes [i.e. the first aspartate-rich motif(FARM) and the second aspartate-rich motif (SARM)]are thought to bind the diphosphate group of the allylicsubstrate via magnesium ions. FARM and SARM arelocated on a-helices D and H. (Note that the presentstudy follows the helix designation first reported for thecrystal structure of avian farnesyl diphosphate synthase(FPS) by Tarsis et al. [4].) Departure of the diphos-phate group forms an allylic carbocation, which isattacked by the p-electron at the double-bond of IPP,forming a new bond between the fourth carbon of IPPand the first carbon of the allylic substrate. Thus, pre-nyl diphosphate is elongated by one C5prenyl unit.The condensation reaction is repeated, elongating theprenyl chain. As the chain elongates, the hydrocarbonmoiety becomes located deep within the reaction cavityformed by a-helices C, D, E, F, G and H. Enzyme-specific termination of prenyl-chain elongation resultsin final products unique to each enzyme.Mutational and structural studies have revealed that,in general, bulky amino acids at the bottom of the cav-ity block prenyl-elongation. In particular, our researchgroup has shown that, in (all-E) prenyl diphosphatesynthases yielding short-chain products such as GGPPand FPP, the bulky amino acids are found in tworegions: upstream from FARM [15] and from thehighly-conserved G(Q/E) motif [16], respectively.FARM exists on a-helix D and the G(Q/E) motif islocated on a-helix F. Based on the characteristicsequences upstream from FARM, the short-chainenzymes have been classified into five types [15]: threetypes of geranylgeranyl diphosphate synthase (GGPS)and two types of FPS (Fig. 1). Type I GGPS fromarchaea has a bulky aromatic amino acid residue,which plays a primary role in the chain length determi-nation mechanism at the fifth position upstream fromFARM. The importance of the residue was shown bymutational studies on GGPS from a thermoacidophilicarchaeon Sulfolobus acidocaldarius [17–19]. TwoGGPSs with known crystal structures [i.e. those froma hyperthermophilic archaeon Pyrococcus horikoshii(Protein Data Bank code 1WY0) and from a thermo-philic bacterium Thermus thermophilus (1WMW)] alsofall into this type. The bulky residue at the fifth posi-tion upstream from FARM is in the center of thecavity and likely to act as the bottom of it in thesestructures; however, the structural information is inde-cisive with respect to the role of the residue becausethe structures do not contain allylic substrates or theiranalogues bound in the active site. Such characteristicFig. 1. Alignment of amino acid sequences around FARM of vari-ous (all-E) prenyl diphosphate synthases. The partial amino acidsequences of the enzymes classified into two types of FPSs synth-ases and three types of GGPSs are aligned. Sce FPS, S. cerevisiaeFPS; Gga FPS, Gallus gallus (avian) FPS; Eco FPS, E. coli FPS; GstFPS, G. stearothermophilus FPS; Sac GGPS, S. acidocaldariusGGPS; Mth GGPS, Methanobacterium thermoautotrophicum GGPS;Pan GGPS, P. ananatis GGPS; Sal GGPS, S. alba (mustard) GGPS;SceGGPS, S. cerevisiae GGPS; Hsa GGPS, Homo sapiens GGPS.The characteristic amino acid residues suggested to be involvedproduct determination for each type of enzyme are shaded.Product determination mechanism of type II GGPS M. Noike et al.3922 FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBSsequences also are evident in FPSs. Eukaryotic type IFPS has bulky amino acids at both the fourth and fifthpositions upstream from FARM. Mutational andstructural studies on avian FPS clarified the role of thepositions [20]. In the structure of mutated avian FPS(1UBX), in which phenylalanine residues at the fourthand fifth positions upstream from FARM are replacedwith serine and alanine, respectively, the x-end of thehydrocarbon chain of FPP bound in the cavity passesthrough the hole formed by the mutagenesis. Broadstructural studies using inhibitor substrate analogueswere made with type 1 FPSs from human [11] and pro-tozoa [6,12], and some of the structures [e.g. FPS fromhuman (2F94 and 3B7L) and Trypanosoma brucei(2P1C and 2I19) binding bisphosphonate inhibitorswith hydrocarbon chains as long as that of GPP] sug-gest the importance of the bulky amino acids becausethey are in contact with the inhibitors. However, nomutational study that supports the hypothesis has beenmade with the enzymes. Bacterial type II FPS has abulky amino acid only at the fifth upstream position,but also has a two amino acid insertion in FARM.Mutational studies of FPS from Geobacillus stearother-mophilus showed that the bulky amino acid upstreamfrom FARM is involved in the chain length determina-tion mechanism [21,22]. The crystal structure of thistype of FPS was elucidated using the enzymes fromseveral bacteria, such as Staphylococcus aureus andEscherichia coli [7]. The structures of E. coli FPS bind-ing substrate analogues (1RQI and 1RQJ) also suggest,but do not ensure, the role of tyrosine at the fifth posi-tion upstream from FARM, which is still distant fromthe analogues with short hydrocarbon chains used inthat study [7]. By contrast, eukaryotic type III GGPS,which lacks bulky amino acids at the fourth or fifthpositions upstream from FARM, was shown to utilizebulky amino acids at the second position upstreamfrom the G(Q/E) motif to terminate chain elongationby our mutational work using GGPS from Saccharo-myces cerevisiae [16]. This information was latersupported by a structural and mutational study on thesame enzyme (2DH4) [9].In the present study, mutational studies of GGPSfrom a bacterial plant pathogen, P. ananatis, wereperformed to investigate the mechanism of chainlength determination in type II GGPS from bacteriaand plants, which has not been identified to date.This type of GGPS lacks bulky aromatic amino acidsat the fourth and fifth positions upstream fromFARM, similar to type III GGPS, whereas it has atwo amino acid insertion in FARM, as does type IIFPS. Unexpectedly, mutations at the fourth and fifthpositions upstream from FARM and at the secondposition upstream from the G(Q/E) motif did notaffect the chain length of the final product. In addi-tion, deletion of the insertion sequence in FARM,which is thought to be involved in the chain lengthdetermination mechanism [18], also had no effect onthe chain length of the final product. An additionalmutational study with type II FPS from G. stearother-mophilus confirmed that the insertion in FARM doesnot play a role in the mechanism of chain lengthdetermination in type II enzymes. These results sug-gest that chain length determination is controlled byanother region of the enzymes. Thus, a new line ofmutants was created based on the crystal structuresof other short-chain enzymes and on the results fromprevious mutational studies. Accordingly, a-helix E,which would be located at the subunit interface ofthe enzyme, was identified as playing a role in theproduct chain length determination mechanism oftype II GGPS. Moreover, this result suggests that theother subunit of the homooligomeric enzyme isinvolved in the product chain length determinationmechanism. This conclusion is supported by therecently-solved crystal structure of type II GGPSfrom mustard [23]. The mechanism of product chainlength determination of type II GGPS identified inthe present study may also explain the participationof noncatalytic subunits in the product determinationmechanisms of some heteromeric enzymes, such asgeranyl diphosphate synthase (GPS) and longer-chainprenyl diphosphate synthases.ResultsRefolding and purification of recombinantP. ananatis GGPSP. ananatis GGPS and the mutant enzymes wereexpressed in E. coli as inclusion bodies. To obtain sol-uble enzymes, inclusion bodies prepared from theinsoluble fraction were denatured by guanidine hydro-chloride and then purified by refolding on a HisTrapcolumn. The purified proteins gave almost single, iden-tical bands by SDS/PAGE (data not shown). Only themutant L128A was completely inactive. All othermutant GGPSs exhibited enzyme activity comparableto that of the wild-type enzyme, whereas L127Ashowed only approximately 20% activity of wild-type.Analysis of the quaternary structure of the refoldedenzyme using blue native PAGE showed that themolecular mass of P. ananatis GGPS is approximately130 or 240 kDa, suggesting that the main part of theenzyme exists as a homotetramer or a homooctamer(Fig. 2).M. Noike et al. Product determination mechanism of type II GGPSFEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3923Mutation in the region upstream from FARMIn type II GGPS, bulky aromatic amino acids at thefourth and/or fifth positions upstream from FARM,which are characteristic of many short-type (all-E) pre-nyl diphosphate synthases, are not present. Previousstudies, mainly conducted by our research group, indi-cated that bulky amino acids on a-helix D, whichincludes FARM, block prenyl-chain elongation,thereby controlling chain length [17–20,22]. To deter-mine whether this mechanism of chain length deter-mination also operates in type II GGPS fromP. ananatis, alanine 89 at the fifth position upstreamfrom FARM was replaced with bulky amino acids(Fig. 3A). These mutations were designed to mimicbacterial type II FPS. The mutants, A89F, A89L andA89H, yielded shorter products than the wild-typeenzyme (Fig. 3B). In particular, the substrate specifici-ties of A89F and A89H were almost identical to thatof FPS: product yield was minimal when GGPP wasused as the substrate. This result suggested that, intype II GGPS, the prenyl-chain of the product elon-gates along a-helix D and that the amino acid residueson a-helix D further upstream from FARM areinvolved in chain length determination. Thus, newmutations were introduced further upstream fromFARM. It was expected that substituting the smalleramino acid, alanine, for the bulky residues wouldincrease the chain length of the final products(Fig. 4A). However, these mutants (i.e., H87A, V86Aand M85A) did not yield longer products than thewild-type (Fig. 4B). H87A activity using GGPP as thesubstrate was undetectable, probably because themutation significantly decreased overall enzyme activ-ity. These results clearly indicated that bulky aminoacids relative to FARM do not contribute to the prod-uct determination mechanism of type II GGPS.Mutation at the second position upstream fromthe G(Q/E) motifBecause the mechanism of chain length determinationfor type II GGPS was shown to be independent fromthe region upstream from FARM, the other regionknown to play a role in chain length determinationwas expected to play a critical role. The second posi-tion upstream from the conserved G(Q/E) motif wasfirst identified as an important residue in the chainlength determination mechanism of type III GGPSfrom S. cerevisiae in a previous study conducted in ourlaboratory [16]. The relatively bulky residue, histidine139, rather than those upstream from FARM, wasfound to block chain-elongation. The role of the resi-due was later supported by Chang et al. [9]: the crystalstructure of S. cerevisiae GGPS that these authorsdetermined demonstrated that histidine 139 forms thebottom of the reaction cavity. Therefore, mutants oftype II GGPS from P. ananatis, in which the residueat the second position upstream from the G(Q/E)Fig. 2. Blue native PAGE of refolded P. ananatis GGPS. The refold-ing procedure is described in the Experimental procedures. Lane 1,molecular mass standard; lane 2, wild-type; lane 3, I121A; lane 4,V125A.ABFig. 3. Introduction of substitutive mutations into the fifth positionupstream from FARM of P. ananatis GGPS. (A) Partial amino acidsequences around FARM of wild-type and mutated enzymes arealigned. The substituted amino acid residues are shaded. (B) TLCautoradiochromatograms of the reaction products of wild-type andmutated enzymes. The products were analyzed as described in theExperimental procedures. The allylic substrate used is indicated atthe top of each autoradiochromatogram. Lane 1, A89F; lane 2,A89L; lane 3, A89H; lane 4, wild-type. Under all assay conditions,< 30% of each substrate reacted. Ori., origin; S.F., solvent front.Product determination mechanism of type II GGPS M. Noike et al.3924 FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBSmotif was replaced with smaller amino acids, were con-structed (Fig. 5A). However, the mutants, V163A,V163G and V163S, did not yield products with a chainlength longer than those produced by the wild-typeenzyme (Fig. 5B). Moreover, some mutants did notgive the C25side-product and exhibited decreased spec-ificity for GGPP. This unexpected result indicated thatthe second position upstream from the G(Q/E) motifdoes not contribute to the mechanism of chain lengthdetermination in type II GGPS. In addition, substi-tution of V163 with a bulky amino acid, phenylala-nine, resulted in loss of activity (data not shown).Deletion of the insertion sequence in FARMOhnuma et al. [18] performed a detailed investigationof the mechanism of chain length determination inshort-chain (all-E) prenyl diphosphate synthase, mainlyusing type I GGPS from S. acidocaldarius to constructvarious mutants. In their study, two amino acids wereinserted in FARM of type I GGPS to mimic type IIFPS because this two amino acid insertion, which isspecifically observed in type II FPS and type II GGPS,was expected to affect the product chain length. TypeI GGPS with the insertion mutation yielded largeramounts of the reaction intermediate, FPP, whereasGGPP remained the final product. Although Ohnumaet al. [18] did not confirm the effect of the insertion byperforming the converse mutation (i.e. deletion of theinsertion from type I FPS or type II GGPS), the inser-tion sequence was thought to play a role in the mecha-nism of chain length determination in type II GGPS.Thus, in the present study, the two amino acids inser-tion was deleted from FARM of type II GGPS fromP. ananatis to confirm the effect of the deletion onproduct chain length (Fig. 6A). However, the mutant,GGPS-DFARM, did not yield a final product with achain length longer than that of the product resultingfrom the wild-type enzyme, which gave a small amountof the C25side-product (Fig. 6C). The mutant enzymeappeared to exhibit reduced activity toward GGPP,although this reduction may have been due to adecrease in overall enzyme activity. This result indi-cates that the insertion does not have a significanteffect on the mechanism of chain length determinationin type II GGPS. In addition, the two amino acidinsertion in FARM was deleted from type II FPSof G. stearothermophilus (Fig. 6B). The mutantFPS-DFARM also showed product specificity similarABFig. 4. Introduction of substitution mutations into the regionupstream from FARM of P. ananatis GGPS. (A) Partial amino acidsequences around FARM of wild-type and mutated enzymes arealigned. The substituted amino acid residues are shaded. (B) TLCautoradiochromatograms of the reaction products of wild-type andmutated enzymes. The products were analyzed as described in theExperimental procedures. The allylic substrate used is indicated atthe top of each autoradiochromatogram. Lane 1, H87A; lane 2,V86A; lane 3, M85A; lane 4, wild-type. Under all assay conditions,< 30% of each substrate reacted. Ori., origin; S.F., solvent front.ABFig. 5. Introduction of substitution mutations into the second posi-tion upstream from the G(Q/E) motif of P. ananatis GGPS. (A) Par-tial amino acid sequences around the G(Q/E) motif of wild-type andmutated enzymes are aligned. The substituted amino acid residuesare shaded. (B) TLC autoradiochromatograms of the reaction prod-ucts of the wild-type and mutated enzymes. The products wereanalyzed as described in the Experimental procedures. The allylicsubstrate used is indicated at the top of each autoradiochromato-gram. Lane 1, V163A; lane 2, V163G; lane 3, V163S; lane 4, wild-type. Under all assay conditions, < 30% of each substrate reacted.Ori., origin; S.F., solvent front.M. Noike et al. Product determination mechanism of type II GGPSFEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3925to that of the wild-type FPS, although the characteris-tics around FARM mimicked those of type I GGPS(Fig. 6D). Therefore, it was concluded that the twoamino acid insertion does not play an important rolein the chain length determination mechanism in eithertype II GGPS or type II FPS.Mutation in a-helix EIn the mutational study on type III GGPS from S. ce-revisiae conducted by Chang et al. [9], the bottom ofthe reaction cavity was suggested to be comprised notonly of histidine 139 at the second position upstreamfrom the G(Q/E) motif, but also of tyrosine 107 andphenylalanine 108. Substituting alanine for tyrosine107 and phenylalanine 108 increased the chain lengthof the final products, as did histidine 139. These bulkyresidues exist in proximity in the structure of theenzyme, which was also reported in the same study.Tyrosine 107 and phenylalanine 108 are located ina-helix E (Chang et al. [9] referred to a-helix E asa-helix F), whereas FARM and the G(Q/E) motif arelocated in a-helices D and F, respectively (D and Gaccording to the designation of Chang et al. [9]).Moreover, the structure of S. cerevisiae GGPS bindingGGPP recently reported (2E8V) revealed that tyrosine107 directly touches the x-end of GGPP bound in thesame subunit, whereas phenylalanine 108 suppliedfrom the other subunit exists in the proximity of thex-end [24].These results led to the hypothesis that the prenyl-chain of the product elongates in the space enclosed bya-helices D, E and F, and that the bulky amino acidresidues on at least one of the a-helices block chain-elongation. If this hypothesis is correct, type II GGPSshould use residues on a-helix E to terminate chain-elongation. Thus, alanine substitution mutations wereintroduced at each position on a-helix E where a bulkyamino acid was located (Fig. 7A). These bulky aminoacids on a-helix E can form the bottom of a reactioncavity similar to those residues located at the key posi-tions upstream from FARM and from the G(Q/E)ABCDFig. 6. Deletion of the insertion sequences in FARM of P. ananatis GGPS and G. stearothermophilus FPS. (A) Partial amino acid sequencesaround FARM of P. ananatis GGPS and mutated enzymes are aligned. The deleted positions are shaded. (B) Partial amino acid sequencesaround FARM of G. stearothermophilus FPS and mutated enzymes are aligned. The deleted positions are shaded. (C) TLC autoradiochroma-tograms of the reaction products of the P. ananatis GGPS and mutated enzymes. The products were analyzed as described in the Experi-mental procedures. The allylic substrate used is indicated at the top of each autoradiochromatogram. Lane 1, GGPS-DFARM; lane 2,wild-type GGPS. Under all assay conditions, < 30% of each substrate reacted. Ori., origin; S.F., solvent front. (D) TLC autoradiochromato-grams of the reaction products of the G. stearothermophilus FPS and mutated enzymes. The products were analyzed as described in theExperimental procedures. The allylic substrate used is indicated at the top of each autoradiochromatogram. Lane 1, FPS-DFARM; lane 2,wild-type FPS. Under all assay conditions, < 30% of each substrate reacted. Ori., origin; S.F., solvent front.Product determination mechanism of type II GGPS M. Noike et al.3926 FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBSmotif in the other types of the enzyme. Among theconstructed mutants, I121A and V125A yielded longerproducts than the wild-type enzyme (Fig. 7B,C). I121Agave a C35product when GGPP was used as the sub-strate, whereas the final product of the wild-typeGGPS was C25prenyl diphosphate. On the otherhand, V125A yielded a series of products whose maxi-mum chain length reached over C40when GPP orGGPP was used as the primer substrate. The othermutants showed product specificity similar to that ofthe wild-type enzyme, although some mutants exhib-ited negligible substrate specificity for GGPP.ABCFig. 7. Introduction of substitutive mutations into the predicted a-helix E of P. ananatis GGPS. (A) Partial amino acid sequences arounda-helix E of wild-type and mutated enzymes are aligned. The substituted amino acid residues are shaded. (B) TLC autoradiochromatogramsof the reaction products of wild-type and mutated enzymes. The products were analyzed as described in the Experimental procedures. Theallylic substrate used is indicated at the top of each autoradiochromatogram. Lane 1, L122A; lane 2, I121A; lane 3, H118A; lane 4, E117A;Lane 5, Y115A; lane 6, H114A; lane 7, wild-type. Under all assay conditions, < 30% of each substrate reacted. Ori., origin; S.F., solventfront. (C) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes. Lane 1, V125A; lane 2, L127A; lane 3,wild-type.M. Noike et al. Product determination mechanism of type II GGPSFEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3927DiscussionIn the present study, type II GGPS from P. ananatiswas recombinantly expressed and purified by refoldingon a column. Blue native PAGE suggested that theenzyme has a homotetrameric or homooctermericstructure. Crystal structural analysis of human GGPSreveals that three homodimers, which comprise thesame quaternary structure observed for most short-chain (all-E) prenyl diphosphate synthases, jointogether to form homohexamer [9]. Thus, in the caseof P. ananatis GGPS, it also is likely that two or fourhomodimers join together to form a homotetramer ora homooctamer, respectively. The recombinant enzymeand its mutants were used to identify amino acid resi-dues that contribute to the mechanism of chain lengthdetermination. Unexpectedly, the two regions that areknown to play important roles in the mechanism insome types of short-chain (all-E) prenyl diphosphatesynthases [i.e. the fourth and fifth positions upstreamfrom FARM and the second position upstream fromthe G(Q/E) motif] were not involved in chain lengthdetermination in type II GGPS. Moreover, a twoamino acid insertion in FARM, which was thought tobe involved in the mechanism of chain length determi-nation, had no significant effect on the product chainlength in either type II GGPS or type II FPS. Alterna-tively, alanine substitution mutations in a-helix Erevealed that isoleucine 121 and valine 125 are the resi-dues involved in the mechanism of chain length deter-mination. To the best of our knowledge, this is thefirst report to describe mutations in type II GGPS thatchange the chain length of the final product of theenzyme.Although it was apparent that a-helix E wasinvolved in the mechanism of chain length determina-tion in type II GGPS, an additional question wasraised. The crystal structures of some of the homodi-meric (all-E) prenyl diphosphate synthases indicatedthat a-helix E exists at the dimer interface. Thus, thequestion arose as to whether the critical residues (i.e.I121 and V125) provided for the reaction cavity arefrom the same catalytic subunit or from the other pair-ing subunit? Fortunately, a crystal structure that wasrecently solved has provided a clear answer to thisquestion. Kloer et al. [23] reported the crystal structureof type II GGPS from mustard (Sinapis alba), bindingGGPP. In the homodimeric structure (2J1P), the gera-nylgeranyl chain of GGPP elongates in the cavityformed by four a-helices, D, E, F (from the catalyticsubunit) and E¢ (from the pairing subunit). V178¢ andD182¢ in a-helix E¢, which correspond to I121 andV125 of P. ananatis GGPS, respectively, exist muchcloser to the geranylgeranyl chain than do V178 andD182 in a-helix E (Fig. 8A). Especially, D182¢ directlytouches the x-end of the geranylgeranyl-chain.Although V178¢ is not in direct contact with GGPP, itappears to support D182¢ or L179¢ at the next positionin a-helix E¢, which touches the center of the geranyl-geranyl-chain and bends it towards the bottom of thecavity formed by L185 in a-helix E, I216 in a-helix F,and D182¢ and S186¢ in a-helix E¢ (Fig. 8B, left). Thefourth and fifth residues upstream from FARM [i.e.S147 and MSE (selenomethionine)146, respectively]also are in contact with the geranylgeranyl chain, butthese residues appear to act only as part of the cavitywall, as does the second residue upstream from theGQ motif (i.e. V222). An almost similar spatialarrangement was observed in the model structure ofABFig. 8. Structural information on the product determination mecha-nism of type II GGPS. (A) The direction of the geranylgeranyl chainof GGPP bound in a subunit (blue) of S. alba GGPS. The x-end ofthe chain touches a-helix E¢ supplied from the other subunit (pink).GGPP is indicated by a cylinder model and some equivalent resi-dues on a-helices E and E¢ are shown as sphere models. (B) Closeview of the substrate pocket of S. alba GGPS (left) and that of themodeled dimeric structure of P. ananatis GGPS (right). The modelof P. ananatis GGPS was constructed based on the crystal struc-ture of S. alba GGPS as the template. Some of the amino acidresidues surrounding the geranylgeranyl chain of GGPP bound inS. alba GGPS and the corresponding residues in P. ananatis GGPSare indicated as sphere models. The geranylgeranyl chain isindicated by green cylinders.Product determination mechanism of type II GGPS M. Noike et al.3928 FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBSP. ananatis GGPS, which was constructed using thecrystal structure of S. alba GGPS as the template formolecular modeling (Fig. 8B, right). From this struc-tural information, it is readily apparent that replace-ment of V178 or D182 of S. alba GGPS with a smalleramino acid would create a new chain-elongation paththrough which the prenyl chain can lengthen acrossthe subunit interface. It is conceivable that a similarscenario also occurred in the I121A and V125Amutants of P. ananatis GGPS.The role of a-helix E in the mechanism of chain lengthdetermination also has been reported for a few short-chain (all-E) prenyl diphosphate synthases of othertypes. As mentioned above, type III GGPS from S. cere-visiae utilizes tyrosine 107 and phenylalanine 108 toterminate elongation of the product [9], although theseresidues do not correspond with the positions 121 and125 of P. ananatis GGPS. The recently reported struc-ture of the complex of S. cerevisiae GGPS and GGPPrevealed that tyrosine 107 was supplied from the othersubunit [24], as is the case for I121 and V125 in type IIGGPS from P. ananatis. On the other hand, Lee et al.[25] reported that, in type II FPS from E. coli, glycinesubstitution of aspartate 115, which also exists in a-helixE, allows the enzyme to produce GGPP. The position ofthe aspartate residue corresponds to V125 in P. ananatisGGPS. Lee et al. [25] hypothesized that the destructionof the hydrogen bond between aspartate 115 and histi-dine 83 increases the flexibility of the a-helices, expand-ing the reaction cavity. In their paper it is likely thatonly the arrangement of the residues in a monomer sub-unit was considered as an explanation of the phenome-non. However, in the crystal structure of E. coli FPSbinding IPP and the analogue of dimethylallyl diphos-phate (DMAPP) (1RQI) [7], aspartate 115 exists in closeproximity to the tyrosine 79¢ at the fifth positionupstream from FARM of the other subunit, which isconsidered to play a significant role in chain lengthdetermination. This observation strongly suggests thatthe aspartate residue might be supplied to offer struc-tural support of tyrosine 79¢ or to block chain-elonga-tion, probably in part, in the other subunit.The results of the present study, which suggest therequirement of subunit interaction for chain lengthdetermination in type II GGPS, are reminiscent of anintriguing study by Burke and Croteau [26]. Theseauthors reported that a subunit of homodimeric typeII GGPS from Taxus candensis and a small subunitof heterotetrameric GPS from Mentha piperita canform a hybrid heterodimer, which yields GPP whenDMAPP is used as the substrate. The large subunit ofM. piperita GPS is very similar to T. candensis GGPS,whereas the small subunit is not. Thus, the small,probably noncatalytic subunit was shown to influencethe product specificity of type II GGPS. It is conceiv-able that the mechanism that acts in P. ananatis GGPSis similar to that observed for the hybrid heteromericenzyme and to the mechanism that likely occurs in het-eromeric GPSs from plants. Heteromeric longer-chain(all-E) prenyl diphosphate synthases have been identi-fied, including heptaprenyl diphosphate (C35) synthasesfrom bacilli [27–29]; hexaprenyl diphosphate (C30) syn-thase from Micrococcus luteus B-P 26 [30]; solanesyldiphosphate (C45) synthase from mouse [31]; and deca-prenyl diphosphate (C50) synthase from human [31]and Schizosaccharomyces pombe [32]. These heteromer-ic enzymes may provide more definitive evidence forsubunit interaction in the mechanism of chain lengthdetermination. Indeed, Zhang et al. [33] reported thatmutation in the small subunit of heterodimeric hepta-prenyl diphsophate synthase from Bacillus subtilis,which shows only slight similarity with homodimeric(all-E) prenyl diphosphate synthases, affects the chainlength of the final product.As noted above, the crystal structures of avian FPSand human GGPS as the complexes with their finalproducts, FPP and GGPP, respectively, have beensolved [10,20]. However, the direction of prenyl-chainelongation differs between these enzymes. In the struc-ture of mutated avian type I FPS binding FPP(1UBX), reported as the monomeric form, the farnesylchain elongates toward the expected dimer interface[20]. Thus, the cavity of avian type I FPS is thought tobe constructed by a-helices D, E, F and probably E¢,as is that of type II GGPS from S. alba. By contrast,in human GGPS binding GGPP (2Q80), the x-end ofthe geranylgeranyl chain enters the space enclosed bya-helices C, D and G [10]. In the structure, the resi-dues known to be important in the chain length deter-mination [i.e. the fourth and fifth positions upstreamfrom FARM and the second position upstream fromthe G(Q/E) motif] just come into contact with theproduct at the center of the prenyl chain, suggestingthat these residues do not act to form the bottom ofthe cavity in human type III GGPS. A similar path ofprenyl-chain elongation was suggested for hexaprenyldiphosphate synthase from Sulfolobus solfataricus.Inamutational study, alanine or glycine substitution forleucine 164 in a-helix G increased the chain length ofthe final product [8]. However, enzymes with chain-elongation paths enclosed by a-helices C, D and Gmight be exceptional because the structural studies ofthe other (all-E) prenyl diphosphate synthases, includ-ing type III GGPS from S. cerevisiae [24] and octapre-nyl diphosphate synthase from Thermotoga maritima[34], as well as a large number of mutational studies,M. Noike et al. Product determination mechanism of type II GGPSFEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3929suggest that the majority of enzymes have pathsenclosed by a-helices D, E and F. In the enzymes pos-sessing structures similar to human GGPS, it is possi-ble that a different type of chain length determinationmechanism exists in which amino acid residues inunknown regions play crucial roles.Experimental proceduresMaterialsPrecoated reversed-phase TLC plates, LKC-18F were pur-chased from Whatman (Maidstone, UK). (all-E) GGPP,(all-E) FPP and (all-E) GPP were synthesized as previouslyreported [35]. Nonlabeled IPP and DMAPP were donatedby C. Ohto (Toyota Motor Co., Japan). [1-14C]IPP waspurchased from GE Healthcare (Piscataway, NJ, USA). Allother chemicals were of analytical grade.General proceduresRestriction enzyme digestions, transformations and otherstandard molecular biology techniques were carried out aspreviously described [36].Plasmid construction and site-directedmutagenesisUsing pACYC-IBE [37], which contains carotenoid biosyn-thetic genes from P. ananatis, as the template, the crtE geneencoding GGPS was amplified using PCR with KOD DNApolymerase (Toyobo, Osaka, Japan) and the primers: PaGGPS-Fw, 5¢-AAGAAACATATGACGGTCTGCGCAAAAAAACACG-3¢, and PaGGPS-Rv, 5¢-TGCAGAGGATCCTTAACTGACGGCAGCGAGTTTTTTG-3¢. The sequen-ces corresponding to the NdeI and BamHI sites that wereused in subsequent experiments are underlined in the pri-mer sequences above. The amplified fragment was cleavedwith the restriction enzymes and then inserted into anNdeI/BamHI-treated pET-15b vector (Novagen, Madison,WI, USA) to construct pET-HisPaGGPS, a plasmid for therecombinant expression of His6-tagged P. ananatis GGPS.For the expression of His6-tagged G. stearothermophilusFPS, the gene was amplified using PCR with KOD DNApolymerase (Toyobo), a pFPS [21], and the primers: His-BsFPS-Fw, 5¢-ACAGCCATGGGACATCATCATCATCATCATGCGCAGCTTTCAGTTGAA-3¢, and HisBsFPS-Rv, 5¢-TGAATTTAAAGCTTAATGGTCGCGGGCG-3¢.The sequences corresponding to the NcoI and HindIII sitesthat were used in subsequent experiments are underlined inthe above sequences. The amplified fragment was cleavedwith the restriction enzymes and then inserted into theNcoI/HindIII-treated pTV118N vector (TaKaRa, Shiga,Japan) to construct pTV-HisBsFPS. Site-directed mutationswere introduced into each parental plasmid utilizing aQuikChange Mutagenesis Kit (Stratagene, La Jolla, CA,USA) according to the manufacturer’s instructions.Expression and purification of wild-type andmutated enzymesFor the expression of P. ananatis GGPS, E. coliBL21(DE3) was transformed with pET-HisPaGGPS or themutated plasmids. The transformants were cultivated in50 mL of M9 minimal broth supplemented with glycerol(2 gÆL)1), yeast extract (2 gÆL)1) and ampicillin (50 mgÆL)1).When D600of 0.5 was reached, the transformed bacteria inthe culture were induced with 1.0 mm isopropyl thio-ß-d-galactoside. The cells were incubated overnight and thenharvested. The cells were disrupted in lysis buffer contain-ing 20 mm sodium phosphate buffer (pH 8.0), 10 mmimidazol and 0.5 m NaCl. The homogenate was centrifugedat 6000 g for 15 min at 4 ° C and the precipitate containingthe inclusion body enzyme was recovered. The precipitatewas dissolved in lysis buffer supplemented with 4%Triton X-100. After shaking for 30 min at 25 °C, themixture was centrifuged at 6000 g for 15 min at 4 °C andthe precipitate was recovered. The process was repeatedtwice to remove bacterial membranous compounds. Thewashed precipitate was lyophilized and used as the inclusionbody. Ten milligrams of the inclusion body was dissolved in10 mL of denaturation buffer containing 50 mm sodiumphosphate buffer (pH 8.0), 10 mm imidazol, 6 m guanidinehydrochloride, 10 mm dithiothreitol, 10 mm 2-mercapto-ethanol and 0.5 m NaCl. After centrifugation at 9000 g for15 min at 4 °C, the supernatant was recovered and thenapplied to a HisTrap column (GE Healthcare) equilibratedwith equilibration buffer containing 50 mm sodiumphosphate buffer (pH 8.0), 10 mm imidazol, 6 m guanidinehydrochloride, 10 mm dithiothreitol, 10 mm 2-mercapto-ethanol and 0.5 m NaCl. The column was washed with10 mL of equilibration buffer and then with start buffer con-taining 50 mm sodium phosphate buffer (pH 8.0), 10 mm im-idazol, 10 mm 2-mercaptoethanol and 0.5 m NaCl to removethe denaturant. The protein renatured in the column waseluted from the column with 10 mL of elution buffer contain-ing 50 mm sodium phosphate buffer (pH 8.0), 500 mm imi-dazol, 10 mm 2-mercaptoethanol and 0.5 m NaCl. The eluatewas fractioned, and the fraction with the highest enzymeactivity and purity was used as the partially purified enzymein the experiments described below. The purity of the enzymewas determined by 15% SDS/PAGE.For the expression of G. stearothermophilus FPS, E. coliDH5a was used as the host. The transformants were culti-vated and induced as described above. Disruption ofharvested cells and the purification of the tagged enzymeswere conducted utilizing a MagExtractor His-tag Kit(Toyobo) according to the manufacturer’s instructions.Product determination mechanism of type II GGPS M. Noike et al.3930 FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS[...]... (2003) An alternative mechanism of product chain- length determination in type III geranylgeranyl diphosphate synthase Eur J Biochem 270, 2186–2194 Ohnuma S-i, Hirooka K, Hemmi H, Ishida C, Ohto C & Nishino T (1996) Conversion of product specificity of archaebacterial geranylgeranyl- diphosphate synthase Identification of essential amino acid residues for chain length determination of prenyltransferase... 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K (1994) Purification and properties of geranylgeranyl- diphosphate synthase from bovine brain J Biol Chem 269, 20561–20566 Kuzuguchi T, Morita Y, Sagami I, Sagami H & Ogura K (1999) Human geranylgeranyl diphosphate synthase cDNA cloning and expression J Biol Chem 274, 5888– 5894 Wang K & Ohnuma S (1999) Chain- length determination mechanism of isoprenyl diphosphate synthases and implications for molecular... chain length determination of prenyltransferases J Biol Chem 273, 26705–26713 Tarshis LC, Proteau PJ, Kellogg BA, Sacchettini JC & Poulter CD (1996) Regulation of product chain length by isoprenyl diphosphate synthases Proc Natl Acad Sci USA 93, 15018–15023 Ohnuma S-i, Nakazawa T, Hemmi H, Hallberg AM, Koyama T, Ogura K & Nishino T (1996) Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate. .. sequences of the genes for two essential proteins constituting a novel enzyme system for heptaprenyl diphosphate synthesis J Biol Chem 270, 18396–18400 28 Zhang YW, Koyama T & Ogura K (1997) Two cistrons of the gerC operon of Bacillus subtilis encode the two subunits of heptaprenyl diphosphate synthase J Bacteriol 179, 1417–1419 29 Zhang YW, Koyama T, Marecak DM, Prestwich GD, Maki Y & Ogura K (1998) Two subunits... 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JE, Bunkoczi G, Russell RG & Oppermann U (2006) The crystal structure of human FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3931 Product determination mechanism of type II GGPS 11 12 13 14 15 16 17 18 19 20 21 M Noike et al geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding J Biol Chem 281, 22004–22012 Kavanagh... diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis J Biol Chem 271, 10087–10095 3932 22 Ohnuma S-i, Narita K, Nakazawa T, Ishida C, Takeuchi Y, Ohto C & Nishino T (1996) A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product J Biol Chem 271, 30748–30754 23 Kloer... Poulter CD & Sacchettini JC (1994) Crystal structure of recombinant farnesyl diphos˚ phate synthase at 2.6-A resolution Biochemistry 33, 10871–10877 5 Guo RT, Kuo CJ, Chou CC, Ko TP, Shr HL, Liang PH & Wang AH (2004) Crystal structure of octaprenyl pyrophosphate synthase from hyperthermophilic Thermotoga maritima and mechanism of product chain length determination J Biol Chem 279, 4903–4912 6 Mao J, Gao . The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction Motoyoshi. that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric
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