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

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction Motoyoshi Noike1,2, Takashi Katagiri1, Toru Nakayama1, Tanetoshi Koyama2, Tokuzo Nishino1 and Hisashi Hemmi1 Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Miyagi, Japan Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi, Japan Keywords farnesyl diphosphate synthase; geranylgeranyl diphosphate synthase; isoprenoid; mutagenesis; prenyltransferase Correspondence H Hemmi, Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan Fax: +81 52 789 4120 Tel: +81 52 789 4134 E-mail: hhemmi@agr.nagoya-u.ac.jp (Received 21 February 2008, revised June 2008, accepted June 2008) doi:10.1111/j.1742-4658.2008.06538.x The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied In most types of short-chain (all-E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl diphosphate synthase Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism However, deletion mutation of the insertion had no effect on product chain length Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the a-helix located at the expected subunit interface were replaced with alanine Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase (All-E) prenyl diphosphate synthase catalyzes the consecutive condensation of isopentenyl diphosphates (IPP) with allylic prenyl diphosphates to yield the final product with a specific prenyl chain length [1,2] The chain length of the product must be tightly controlled because polymerization of isoprene units is the key reaction responsible for the tremendous variety of naturally occurring isoprenoid compounds (> 50 000) [3] For example, many important compounds, such as carotenoids, tocopherols, diterpenes, chrolophyll and archaeal membrane lipids, are synthesized from geranylgeranyl diphosphate (GGPP; C20) On the other Abbreviations DMAPP, 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 3921 Product determination mechanism of type II GGPS M Noike et al hand, farnesyl diphosphate (FPP; C15) is the precursor of steroids, sesquiterpenes and heme a Moreover, FPP is the usual allyic primer substrate for prenyl elongation reactions, which yield longer-chain prenyl diphosphates as the precursors of respiratory quinones, dolichol and natural rubber, although some organisms also use GGPP for the same purpose Longer-chain (all-E) prenyl diphosphates (up to C60) are utilized for the biosynthesis of various respiratory quinones, which have been used to classify the microorganisms GGPP and FPP are also utilized for protein modification, although they modify very different classes of acceptor proteins Rab family G proteins are geranylgeranylated, whereas farnesylation typically occurs on Ras proteins In addition, geranyl diphosphate (GPP; C10) is the precursor of volatile monoterpenes and also is used to modify secondary metabolites The mechanism of prenyl-chain elongation, and therefore of product determination in (all-E) prenyl diphosphate synthases, which share many conserved sequences in spite of their different reaction products, has been investigated previously The enzymes are constructed mainly of a-helices, which form a large reaction cavity per a subunit [4–12] Most of the enzymes are homodimeric proteins, although some enzymes consist of heterodimers with little homology between the subunits [1] A few mammalian enzymes are known to form oligomers [13,14] The highly conserved motifs of 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 allylic substrate via magnesium ions FARM and SARM are located on a-helices D and H (Note that the present study follows the helix designation first reported for the crystal structure of avian farnesyl diphosphate synthase (FPS) by Tarsis et al [4].) Departure of the diphosphate group forms an allylic carbocation, which is attacked by the p-electron at the double-bond of IPP, forming a new bond between the fourth carbon of IPP and the first carbon of the allylic substrate Thus, prenyl diphosphate is elongated by one C5 prenyl unit The condensation reaction is repeated, elongating the prenyl chain As the chain elongates, the hydrocarbon moiety becomes located deep within the reaction cavity formed by a-helices C, D, E, F, G and H Enzymespecific termination of prenyl-chain elongation results in final products unique to each enzyme Mutational and structural studies have revealed that, in general, bulky amino acids at the bottom of the cavity block prenyl-elongation In particular, our research group has shown that, in (all-E) prenyl diphosphate synthases yielding short-chain products such as GGPP and FPP, the bulky amino acids are found in two 3922 regions: upstream from FARM [15] and from the highly-conserved G(Q/E) motif [16], respectively FARM exists on a-helix D and the G(Q/E) motif is located on a-helix F Based on the characteristic sequences upstream from FARM, the short-chain enzymes have been classified into five types [15]: three types of geranylgeranyl diphosphate synthase (GGPS) and two types of FPS (Fig 1) Type I GGPS from archaea has a bulky aromatic amino acid residue, which plays a primary role in the chain length determination mechanism at the fifth position upstream from FARM The importance of the residue was shown by mutational studies on GGPS from a thermoacidophilic archaeon Sulfolobus acidocaldarius [17–19] Two GGPSs with known crystal structures [i.e those from a hyperthermophilic archaeon Pyrococcus horikoshii (Protein Data Bank code 1WY0) and from a thermophilic bacterium Thermus thermophilus (1WMW)] also fall into this type The bulky residue at the fifth position upstream from FARM is in the center of the cavity and likely to act as the bottom of it in these structures; however, the structural information is indecisive with respect to the role of the residue because the structures not contain allylic substrates or their analogues bound in the active site Such characteristic Fig Alignment of amino acid sequences around FARM of various (all-E) prenyl diphosphate synthases The partial amino acid sequences of the enzymes classified into two types of FPSs synthases and three types of GGPSs are aligned Sce FPS, S cerevisiae FPS; Gga FPS, Gallus gallus (avian) FPS; Eco FPS, E coli FPS; Gst FPS, G stearothermophilus FPS; Sac GGPS, S acidocaldarius GGPS; 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 involved product determination for each type of enzyme are shaded FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS M Noike et al sequences also are evident in FPSs Eukaryotic type I FPS has bulky amino acids at both the fourth and fifth positions upstream from FARM Mutational and structural studies on avian FPS clarified the role of the positions [20] In the structure of mutated avian FPS (1UBX), in which phenylalanine residues at the fourth and fifth positions upstream from FARM are replaced with serine and alanine, respectively, the x-end of the hydrocarbon chain of FPP bound in the cavity passes through the hole formed by the mutagenesis Broad structural studies using inhibitor substrate analogues were made with type FPSs from human [11] and protozoa [6,12], and some of the structures [e.g FPS from human (2F94 and 3B7L) and Trypanosoma brucei (2P1C and 2I19) binding bisphosphonate inhibitors with hydrocarbon chains as long as that of GPP] suggest the importance of the bulky amino acids because they are in contact with the inhibitors However, no mutational study that supports the hypothesis has been made with the enzymes Bacterial type II FPS has a bulky amino acid only at the fifth upstream position, but also has a two amino acid insertion in FARM Mutational studies of FPS from Geobacillus stearothermophilus showed that the bulky amino acid upstream from FARM is involved in the chain length determination mechanism [21,22] The crystal structure of this type of FPS was elucidated using the enzymes from several bacteria, such as Staphylococcus aureus and Escherichia coli [7] The structures of E coli FPS binding substrate analogues (1RQI and 1RQJ) also suggest, but not ensure, the role of tyrosine at the fifth position upstream from FARM, which is still distant from the analogues with short hydrocarbon chains used in that study [7] By contrast, eukaryotic type III GGPS, which lacks bulky amino acids at the fourth or fifth positions upstream from FARM, was shown to utilize bulky amino acids at the second position upstream from the G(Q/E) motif to terminate chain elongation by our mutational work using GGPS from Saccharomyces cerevisiae [16] This information was later supported by a structural and mutational study on the same enzyme (2DH4) [9] In the present study, mutational studies of GGPS from a bacterial plant pathogen, P ananatis, were performed to investigate the mechanism of chain length determination in type II GGPS from bacteria and plants, which has not been identified to date This type of GGPS lacks bulky aromatic amino acids at the fourth and fifth positions upstream from FARM, similar to type III GGPS, whereas it has a two amino acid insertion in FARM, as does type II FPS Unexpectedly, mutations at the fourth and fifth positions upstream from FARM and at the second Product determination mechanism of type II GGPS position upstream from the G(Q/E) motif did not affect the chain length of the final product In addition, deletion of the insertion sequence in FARM, which is thought to be involved in the chain length determination mechanism [18], also had no effect on the chain length of the final product An additional mutational study with type II FPS from G stearothermophilus confirmed that the insertion in FARM does not play a role in the mechanism of chain length determination in type II enzymes These results suggest that chain length determination is controlled by another region of the enzymes Thus, a new line of mutants was created based on the crystal structures of other short-chain enzymes and on the results from previous mutational studies Accordingly, a-helix E, which would be located at the subunit interface of the enzyme, was identified as playing a role in the product chain length determination mechanism of type II GGPS Moreover, this result suggests that the other subunit of the homooligomeric enzyme is involved in the product chain length determination mechanism This conclusion is supported by the recently-solved crystal structure of type II GGPS from mustard [23] The mechanism of product chain length determination of type II GGPS identified in the present study may also explain the participation of noncatalytic subunits in the product determination mechanisms of some heteromeric enzymes, such as geranyl diphosphate synthase (GPS) and longer-chain prenyl diphosphate synthases Results Refolding and purification of recombinant P ananatis GGPS P ananatis GGPS and the mutant enzymes were expressed in E coli as inclusion bodies To obtain soluble enzymes, inclusion bodies prepared from the insoluble fraction were denatured by guanidine hydrochloride and then purified by refolding on a HisTrap column The purified proteins gave almost single, identical bands by SDS/PAGE (data not shown) Only the mutant L128A was completely inactive All other mutant GGPSs exhibited enzyme activity comparable to that of the wild-type enzyme, whereas L127A showed only approximately 20% activity of wild-type Analysis of the quaternary structure of the refolded enzyme using blue native PAGE showed that the molecular mass of P ananatis GGPS is approximately 130 or 240 kDa, suggesting that the main part of the enzyme exists as a homotetramer or a homooctamer (Fig 2) FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3923 Product determination mechanism of type II GGPS M Noike et al A B Fig Blue native PAGE of refolded P ananatis GGPS The refolding procedure is described in the Experimental procedures Lane 1, molecular mass standard; lane 2, wild-type; lane 3, I121A; lane 4, V125A Mutation in the region upstream from FARM In type II GGPS, bulky aromatic amino acids at the fourth and/or fifth positions upstream from FARM, which are characteristic of many short-type (all-E) prenyl diphosphate synthases, are not present Previous studies, mainly conducted by our research group, indicated that bulky amino acids on a-helix D, which includes FARM, block prenyl-chain elongation, thereby controlling chain length [17–20,22] To determine whether this mechanism of chain length determination also operates in type II GGPS from P ananatis, alanine 89 at the fifth position upstream from FARM was replaced with bulky amino acids (Fig 3A) These mutations were designed to mimic bacterial type II FPS The mutants, A89F, A89L and A89H, yielded shorter products than the wild-type enzyme (Fig 3B) In particular, the substrate specificities of A89F and A89H were almost identical to that of FPS: product yield was minimal when GGPP was used as the substrate This result suggested that, in type II GGPS, the prenyl-chain of the product elongates along a-helix D and that the amino acid residues on a-helix D further upstream from FARM are involved in chain length determination Thus, new mutations were introduced further upstream from FARM It was expected that substituting the smaller amino acid, alanine, for the bulky residues would increase the chain length of the final products (Fig 4A) However, these mutants (i.e., H87A, V86A and M85A) did not yield longer products than the wild-type (Fig 4B) H87A activity using GGPP as the substrate was undetectable, probably because the mutation significantly decreased overall enzyme activity These results clearly indicated that bulky amino 3924 Fig Introduction of substitutive mutations into the fifth position upstream from FARM of P ananatis GGPS (A) Partial amino acid sequences around FARM of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the 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 acids relative to FARM not contribute to the product determination mechanism of type II GGPS Mutation at the second position upstream from the G(Q/E) motif Because the mechanism of chain length determination for type II GGPS was shown to be independent from the region upstream from FARM, the other region known to play a role in chain length determination was expected to play a critical role The second position upstream from the conserved G(Q/E) motif was first identified as an important residue in the chain length determination mechanism of type III GGPS from S cerevisiae in a previous study conducted in our laboratory [16] The relatively bulky residue, histidine 139, rather than those upstream from FARM, was found to block chain-elongation The role of the residue was later supported by Chang et al [9]: the crystal structure of S cerevisiae GGPS that these authors determined demonstrated that histidine 139 forms the bottom of the reaction cavity Therefore, mutants of type II GGPS from P ananatis, in which the residue at the second position upstream from the G(Q/E) FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS M Noike et al Product determination mechanism of type II GGPS A A B B Fig Introduction of substitution mutations into the region upstream from FARM of P ananatis GGPS (A) Partial amino acid sequences around FARM of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the 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 Fig Introduction of substitution mutations into the second position upstream from the G(Q/E) motif of P ananatis GGPS (A) Partial amino acid sequences around the G(Q/E) motif of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms of the reaction products of the wild-type and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the top of each autoradiochromatogram Lane 1, V163A; lane 2, V163G; lane 3, V163S; lane 4, wildtype Under all assay conditions, < 30% of each substrate reacted Ori., origin; S.F., solvent front motif was replaced with smaller amino acids, were constructed (Fig 5A) However, the mutants, V163A, V163G and V163S, did not yield products with a chain length longer than those produced by the wild-type enzyme (Fig 5B) Moreover, some mutants did not give the C25 side-product and exhibited decreased specificity for GGPP This unexpected result indicated that the second position upstream from the G(Q/E) motif does not contribute to the mechanism of chain length determination in type II GGPS In addition, substitution of V163 with a bulky amino acid, phenylalanine, resulted in loss of activity (data not shown) Deletion of the insertion sequence in FARM Ohnuma et al [18] performed a detailed investigation of the mechanism of chain length determination in short-chain (all-E) prenyl diphosphate synthase, mainly using type I GGPS from S acidocaldarius to construct various mutants In their study, two amino acids were inserted in FARM of type I GGPS to mimic type II FPS because this two amino acid insertion, which is specifically observed in type II FPS and type II GGPS, was expected to affect the product chain length Type I GGPS with the insertion mutation yielded larger amounts of the reaction intermediate, FPP, whereas GGPP remained the final product Although Ohnuma et al [18] did not confirm the effect of the insertion by performing the converse mutation (i.e deletion of the insertion from type I FPS or type II GGPS), the insertion sequence was thought to play a role in the mechanism of chain length determination in type II GGPS Thus, in the present study, the two amino acids insertion was deleted from FARM of type II GGPS from P ananatis to confirm the effect of the deletion on product chain length (Fig 6A) However, the mutant, GGPS-DFARM, did not yield a final product with a chain length longer than that of the product resulting from the wild-type enzyme, which gave a small amount of the C25 side-product (Fig 6C) The mutant enzyme appeared to exhibit reduced activity toward GGPP, although this reduction may have been due to a decrease in overall enzyme activity This result indicates that the insertion does not have a significant effect on the mechanism of chain length determination in type II GGPS In addition, the two amino acid insertion in FARM was deleted from type II FPS of G stearothermophilus (Fig 6B) The mutant FPS-DFARM also showed product specificity similar FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3925 Product determination mechanism of type II GGPS M Noike et al A B C D Fig Deletion of the insertion sequences in FARM of P ananatis GGPS and G stearothermophilus FPS (A) Partial amino acid sequences around FARM of P ananatis GGPS and mutated enzymes are aligned The deleted positions are shaded (B) Partial amino acid sequences around FARM of G stearothermophilus FPS and mutated enzymes are aligned The deleted positions are shaded (C) TLC autoradiochromatograms of the reaction products of the P ananatis GGPS and mutated enzymes The products were analyzed as described in the Experimental 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 autoradiochromatograms of the reaction products of the G stearothermophilus FPS and mutated enzymes The products were analyzed as described in the Experimental 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 to that of the wild-type FPS, although the characteristics around FARM mimicked those of type I GGPS (Fig 6D) Therefore, it was concluded that the two amino acid insertion does not play an important role in the chain length determination mechanism in either type II GGPS or type II FPS Mutation in a-helix E In the mutational study on type III GGPS from S cerevisiae conducted by Chang et al [9], the bottom of the reaction cavity was suggested to be comprised not only of histidine 139 at the second position upstream from the G(Q/E) motif, but also of tyrosine 107 and phenylalanine 108 Substituting alanine for tyrosine 107 and phenylalanine 108 increased the chain length of the final products, as did histidine 139 These bulky residues exist in proximity in the structure of the enzyme, which was also reported in the same study Tyrosine 107 and phenylalanine 108 are located in a-helix E (Chang et al [9] referred to a-helix E as 3926 a-helix F), whereas FARM and the G(Q/E) motif are located in a-helices D and F, respectively (D and G according to the designation of Chang et al [9]) Moreover, the structure of S cerevisiae GGPS binding GGPP recently reported (2E8V) revealed that tyrosine 107 directly touches the x-end of GGPP bound in the same subunit, whereas phenylalanine 108 supplied from the other subunit exists in the proximity of the x-end [24] These results led to the hypothesis that the prenylchain of the product elongates in the space enclosed by a-helices D, E and F, and that the bulky amino acid residues on at least one of the a-helices block chainelongation If this hypothesis is correct, type II GGPS should use residues on a-helix E to terminate chainelongation Thus, alanine substitution mutations were introduced at each position on a-helix E where a bulky amino acid was located (Fig 7A) These bulky amino acids on a-helix E can form the bottom of a reaction cavity similar to those residues located at the key positions upstream from FARM and from the G(Q/E) FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS M Noike et al Product determination mechanism of type II GGPS A B C Fig Introduction of substitutive mutations into the predicted a-helix E of P ananatis GGPS (A) Partial amino acid sequences around a-helix E of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic 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., solvent front (C) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes Lane 1, V125A; lane 2, L127A; lane 3, wild-type motif in the other types of the enzyme Among the constructed mutants, I121A and V125A yielded longer products than the wild-type enzyme (Fig 7B,C) I121A gave a C35 product when GGPP was used as the substrate, whereas the final product of the wild-type GGPS was C25 prenyl diphosphate On the other hand, V125A yielded a series of products whose maximum chain length reached over C40 when GPP or GGPP was used as the primer substrate The other mutants showed product specificity similar to that of the wild-type enzyme, although some mutants exhibited negligible substrate specificity for GGPP FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3927 Product determination mechanism of type II GGPS M Noike et al Discussion In the present study, type II GGPS from P ananatis was recombinantly expressed and purified by refolding on a column Blue native PAGE suggested that the enzyme has a homotetrameric or homooctermeric structure Crystal structural analysis of human GGPS reveals that three homodimers, which comprise the same quaternary structure observed for most shortchain (all-E) prenyl diphosphate synthases, join together to form homohexamer [9] Thus, in the case of P ananatis GGPS, it also is likely that two or four homodimers join together to form a homotetramer or a homooctamer, respectively The recombinant enzyme and its mutants were used to identify amino acid residues that contribute to the mechanism of chain length determination Unexpectedly, the two regions that are known to play important roles in the mechanism in some types of short-chain (all-E) prenyl diphosphate synthases [i.e the fourth and fifth positions upstream from FARM and the second position upstream from the G(Q/E) motif] were not involved in chain length determination in type II GGPS Moreover, a two amino acid insertion in FARM, which was thought to be involved in the mechanism of chain length determination, had no significant effect on the product chain length in either type II GGPS or type II FPS Alternatively, alanine substitution mutations in a-helix E revealed that isoleucine 121 and valine 125 are the residues involved in the mechanism of chain length determination To the best of our knowledge, this is the first report to describe mutations in type II GGPS that change the chain length of the final product of the enzyme Although it was apparent that a-helix E was involved in the mechanism of chain length determination in type II GGPS, an additional question was raised The crystal structures of some of the homodimeric (all-E) prenyl diphosphate synthases indicated that a-helix E exists at the dimer interface Thus, the question arose as to whether the critical residues (i.e I121 and V125) provided for the reaction cavity are from the same catalytic subunit or from the other pairing subunit? Fortunately, a crystal structure that was recently solved has provided a clear answer to this question Kloer et al [23] reported the crystal structure of type II GGPS from mustard (Sinapis alba), binding GGPP In the homodimeric structure (2J1P), the geranylgeranyl chain of GGPP elongates in the cavity formed by four a-helices, D, E, F (from the catalytic subunit) and E¢ (from the pairing subunit) V178¢ and D182¢ in a-helix E¢, which correspond to I121 and V125 of P ananatis GGPS, respectively, exist much 3928 closer to the geranylgeranyl chain than V178 and D182 in a-helix E (Fig 8A) Especially, D182¢ directly touches the x-end of the geranylgeranyl-chain Although V178¢ is not in direct contact with GGPP, it appears to support D182¢ or L179¢ at the next position in a-helix E¢, which touches the center of the geranylgeranyl-chain and bends it towards the bottom of the cavity formed by L185 in a-helix E, I216 in a-helix F, and D182¢ and S186¢ in a-helix E¢ (Fig 8B, left) The fourth and fifth residues upstream from FARM [i.e S147 and MSE (selenomethionine)146, respectively] also are in contact with the geranylgeranyl chain, but these residues appear to act only as part of the cavity wall, as does the second residue upstream from the GQ motif (i.e V222) An almost similar spatial arrangement was observed in the model structure of A B Fig Structural information on the product determination mechanism of type II GGPS (A) The direction of the geranylgeranyl chain of GGPP bound in a subunit (blue) of S alba GGPS The x-end of the chain touches a-helix E¢ supplied from the other subunit (pink) GGPP is indicated by a cylinder model and some equivalent residues on a-helices E and E¢ are shown as sphere models (B) Close view of the substrate pocket of S alba GGPS (left) and that of the modeled dimeric structure of P ananatis GGPS (right) The model of P ananatis GGPS was constructed based on the crystal structure of S alba GGPS as the template Some of the amino acid residues surrounding the geranylgeranyl chain of GGPP bound in S alba GGPS and the corresponding residues in P ananatis GGPS are indicated as sphere models The geranylgeranyl chain is indicated by green cylinders FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS M Noike et al P ananatis GGPS, which was constructed using the crystal structure of S alba GGPS as the template for molecular modeling (Fig 8B, right) From this structural information, it is readily apparent that replacement of V178 or D182 of S alba GGPS with a smaller amino acid would create a new chain-elongation path through which the prenyl chain can lengthen across the subunit interface It is conceivable that a similar scenario also occurred in the I121A and V125A mutants of P ananatis GGPS The role of a-helix E in the mechanism of chain length determination also has been reported for a few shortchain (all-E) prenyl diphosphate synthases of other types As mentioned above, type III GGPS from S cerevisiae utilizes tyrosine 107 and phenylalanine 108 to terminate elongation of the product [9], although these residues not correspond with the positions 121 and 125 of P ananatis GGPS The recently reported structure of the complex of S cerevisiae GGPS and GGPP revealed that tyrosine 107 was supplied from the other subunit [24], as is the case for I121 and V125 in type II GGPS from P ananatis On the other hand, Lee et al [25] reported that, in type II FPS from E coli, glycine substitution of aspartate 115, which also exists in a-helix E, allows the enzyme to produce GGPP The position of the aspartate residue corresponds to V125 in P ananatis GGPS Lee et al [25] hypothesized that the destruction of the hydrogen bond between aspartate 115 and histidine 83 increases the flexibility of the a-helices, expanding the reaction cavity In their paper it is likely that only the arrangement of the residues in a monomer subunit was considered as an explanation of the phenomenon However, in the crystal structure of E coli FPS binding IPP and the analogue of dimethylallyl diphosphate (DMAPP) (1RQI) [7], aspartate 115 exists in close proximity to the tyrosine 79¢ at the fifth position upstream from FARM of the other subunit, which is considered to play a significant role in chain length determination This observation strongly suggests that the aspartate residue might be supplied to offer structural support of tyrosine 79¢ or to block chain-elongation, probably in part, in the other subunit The results of the present study, which suggest the requirement of subunit interaction for chain length determination in type II GGPS, are reminiscent of an intriguing study by Burke and Croteau [26] These authors reported that a subunit of homodimeric type II GGPS from Taxus candensis and a small subunit of heterotetrameric GPS from Mentha piperita can form a hybrid heterodimer, which yields GPP when DMAPP is used as the substrate The large subunit of M piperita GPS is very similar to T candensis GGPS, whereas the small subunit is not Thus, the small, Product determination mechanism of type II GGPS probably noncatalytic subunit was shown to influence the product specificity of type II GGPS It is conceivable that the mechanism that acts in P ananatis GGPS is similar to that observed for the hybrid heteromeric enzyme and to the mechanism that likely occurs in heteromeric GPSs from plants Heteromeric longer-chain (all-E) prenyl diphosphate synthases have been identified, including heptaprenyl diphosphate (C35) synthases from bacilli [27–29]; hexaprenyl diphosphate (C30) synthase from Micrococcus luteus B-P 26 [30]; solanesyl diphosphate (C45) synthase from mouse [31]; and decaprenyl diphosphate (C50) synthase from human [31] and Schizosaccharomyces pombe [32] These heteromeric enzymes may provide more definitive evidence for subunit interaction in the mechanism of chain length determination Indeed, Zhang et al [33] reported that mutation in the small subunit of heterodimeric heptaprenyl diphsophate synthase from Bacillus subtilis, which shows only slight similarity with homodimeric (all-E) prenyl diphosphate synthases, affects the chain length of the final product As noted above, the crystal structures of avian FPS and human GGPS as the complexes with their final products, FPP and GGPP, respectively, have been solved [10,20] However, the direction of prenyl-chain elongation differs between these enzymes In the structure of mutated avian type I FPS binding FPP (1UBX), reported as the monomeric form, the farnesyl chain elongates toward the expected dimer interface [20] Thus, the cavity of avian type I FPS is thought to be 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 of the geranylgeranyl chain enters the space enclosed by a-helices C, D and G [10] In the structure, the residues known to be important in the chain length determination [i.e the fourth and fifth positions upstream from FARM and the second position upstream from the G(Q/E) motif] just come into contact with the product at the center of the prenyl chain, suggesting that these residues not act to form the bottom of the cavity in human type III GGPS A similar path of prenyl-chain elongation was suggested for hexaprenyl diphosphate synthase from Sulfolobus solfataricus In a mutational study, alanine or glycine substitution for leucine 164 in a-helix G increased the chain length of the final product [8] However, enzymes with chainelongation paths enclosed by a-helices C, D and G might be exceptional because the structural studies of the other (all-E) prenyl diphosphate synthases, including type III GGPS from S cerevisiae [24] and octaprenyl diphosphate synthase from Thermotoga maritima [34], as well as a large number of mutational studies, FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3929 Product determination mechanism of type II GGPS M Noike et al suggest that the majority of enzymes have paths enclosed by a-helices D, E and F In the enzymes possessing structures similar to human GGPS, it is possible that a different type of chain length determination mechanism exists in which amino acid residues in unknown regions play crucial roles Experimental procedures Materials Precoated reversed-phase TLC plates, LKC-18F were purchased from Whatman (Maidstone, UK) (all-E) GGPP, (all-E) FPP and (all-E) GPP were synthesized as previously reported [35] Nonlabeled IPP and DMAPP were donated by C Ohto (Toyota Motor Co., Japan) [1-14C]IPP was purchased from GE Healthcare (Piscataway, NJ, USA) All other chemicals were of analytical grade General procedures Restriction enzyme digestions, transformations and other standard molecular biology techniques were carried out as previously described [36] Plasmid construction and site-directed mutagenesis Using pACYC-IBE [37], which contains carotenoid biosynthetic genes from P ananatis, as the template, the crtE gene encoding GGPS was amplified using PCR with KOD DNA polymerase (Toyobo, Osaka, Japan) and the primers: PaG GPS-Fw, 5¢-AAGAAACATATGACGGTCTGCGCAAA AAAACACG-3¢, and PaGGPS-Rv, 5¢-TGCAGAGGATCC TTAACTGACGGCAGCGAGTTTTTTG-3¢ The sequences corresponding to the NdeI and BamHI sites that were used in subsequent experiments are underlined in the primer sequences above The amplified fragment was cleaved with the restriction enzymes and then inserted into an NdeI/BamHI-treated pET-15b vector (Novagen, Madison, WI, USA) to construct pET-HisPaGGPS, a plasmid for the recombinant expression of His6-tagged P ananatis GGPS For the expression of His6-tagged G stearothermophilus FPS, the gene was amplified using PCR with KOD DNA polymerase (Toyobo), a pFPS [21], and the primers: HisBsFPS-Fw, 5¢-ACAGCCATGGGACATCATCATCATCA TCATGCGCAGCTTTCAGTTGAA-3¢, and HisBsFPSRv, 5¢-TGAATTTAAAGCTTAATGGTCGCGGGCG-3¢ The sequences corresponding to the NcoI and HindIII sites that were used in subsequent experiments are underlined in the above sequences The amplified fragment was cleaved with the restriction enzymes and then inserted into the NcoI/HindIII-treated pTV118N vector (TaKaRa, Shiga, Japan) to construct pTV-HisBsFPS Site-directed mutations 3930 were introduced into each parental plasmid utilizing a QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions Expression and purification of wild-type and mutated enzymes For the expression of P ananatis GGPS, E coli BL21(DE3) was transformed with pET-HisPaGGPS or the mutated plasmids The transformants were cultivated in 50 mL of M9 minimal broth supplemented with glycerol (2 gỈL)1), yeast extract (2 gỈL)1) and ampicillin (50 mgỈL)1) When D600 of 0.5 was reached, the transformed bacteria in the culture were induced with 1.0 mm isopropyl thio-ß-dgalactoside The cells were incubated overnight and then harvested The cells were disrupted in lysis buffer containing 20 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol and 0.5 m NaCl The homogenate was centrifuged at 6000 g for 15 at °C and the precipitate containing the inclusion body enzyme was recovered The precipitate was dissolved in lysis buffer supplemented with 4% Triton X-100 After shaking for 30 at 25 °C, the mixture was centrifuged at 6000 g for 15 at °C and the precipitate was recovered The process was repeated twice to remove bacterial membranous compounds The washed precipitate was lyophilized and used as the inclusion body Ten milligrams of the inclusion body was dissolved in 10 mL of denaturation buffer containing 50 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol, m guanidine hydrochloride, 10 mm dithiothreitol, 10 mm 2-mercaptoethanol and 0.5 m NaCl After centrifugation at 9000 g for 15 at °C, the supernatant was recovered and then applied to a HisTrap column (GE Healthcare) equilibrated with equilibration buffer containing 50 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol, m guanidine hydrochloride, 10 mm dithiothreitol, 10 mm 2-mercaptoethanol and 0.5 m NaCl The column was washed with 10 mL of equilibration buffer and then with start buffer containing 50 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol, 10 mm 2-mercaptoethanol and 0.5 m NaCl to remove the denaturant The protein renatured in the column was eluted from the column with 10 mL of elution buffer containing 50 mm sodium phosphate buffer (pH 8.0), 500 mm imidazol, 10 mm 2-mercaptoethanol and 0.5 m NaCl The eluate was fractioned, and the fraction with the highest enzyme activity and purity was used as the partially purified enzyme in the experiments described below The purity of the enzyme was determined by 15% SDS/PAGE For the expression of G stearothermophilus FPS, E coli DH5a was used as the host The transformants were cultivated and induced as described above Disruption of harvested cells and the purification of the tagged enzymes were conducted utilizing a MagExtractor His-tag Kit (Toyobo) according to the manufacturer’s instructions FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS M Noike et al Enzyme purify was determined by 15% SDS/PAGE (data not shown) Analysis of quaternary structure of refolded P ananatis GGPS The quaternary structures of refolded wild-type and mutated P ananatis GGPSs were analyzed by blue native PAGE [38] using a NativePAGEÔ NovexÒ 4–16% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) The cathode buffer was composed of 50 mm BisTris, 50 mm Tricine (pH 6.8) and 0.02% Coomassie Brilliant Blue G 250, whereas the anode buffer contained 50 mm BisTris (pH 6.8) Product determination mechanism of type II GGPS dimeric S alba GGPS binding a molecule of GGPP (2J1P) as the template pymol (http://www.pymol.org) and imol (http://www.pirx.com/iMol) were used to generate figures from the known structure and constructed model Acknowledgements This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan We are grateful to Dr Chikara Ohto, Toyota Motor Co., for providing isopentenyl diphosphate and dimethylallyl diphosphate References Measurement of prenyltransferase activity The assay mixture for wild-type and mutant P ananatis GGPSs contained, in a final volume of 200 lL, 0.5 nmol of [1-14C]IPP (2 GBqỈmmol)1), 0.5 nmol of an allylic primer (GPP, FPP or GGPP), lmol of MgCl2, lmol of a Tris– HCl buffer (pH 8.0) and a suitable amount of each enzyme This mixture was incubated at 30 °C for 15 and the reaction was stopped by chilling the mixture in an ice bath The mixture was shaken with 600 lL of 1-butanol saturated with H2O The butanol layer was washed with water saturated with NaCl, and the radioactivity in 100 lL of the butanol layer was measured with a TRI-CARB 1600 liquid scintilation counter (Packard Instrument Company, Meriden, CT, USA) The remaining butanol layer was used for product analysis For the assay of G stearothrmophilus FPS, the reaction mixture contained nmol [1-14C]IPP (2 GBqỈnmol)1), nmol allylic primer (DMAPP or GPP), lmol MgCl2, lmol Tris/HCl buffer (pH 8.5), 10 lmol ammonium chloride, 10 lmol 2-mercaptoethanol and a suitable amount of each enzyme in a final volume of 200 lL The mixture was incubated at 55 °C for 10 and then processed as described above Product analysis Prenyl diphosphates in the residual 1-butanol layer were treated with acid phosphatase according to the method of Fujii et al [39] The hydrolysates were extracted with pentane and analyzed by reversed-phase TLC using a precoated plate, LKC-18F, developed with acetone/H2O (9 : 1) Authentic standard alcohols were visualized with iodine vapor, and the distribution of radioactivity was detected using a Molecular imager (Bio-Rad, Hercules, CA, USA) or a BAS 1000 Mac bioimaging analyzer (Fujifilm, Tokyo, Japan) Homology modeling The 3D model of dimeric P ananatis GGPS was constructed with MODELLER [40], using the structure of Ogura K & Koyama T 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Wittig I, Braun HP & Schagger H (2006) Blue native PAGE Nat Protoc 1, 418–428 Fujii H, Koyama T & Ogura K (1982) Efficient enzymatic hydrolysis of polyprenyl pyrophosphates Biochim Biophys Acta 712, 716–718 Sali A & Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints J Mol Biol 234, 779–815 FEBS Journal 275 (2008) 3921–3933 ª 2008 The Authors Journal compilation ª 2008 FEBS 3933 ... in the mechanism of chain length determination To the best of our knowledge, this is the first report to describe mutations in type II GGPS that change the chain length of the final product of the. .. [23] The mechanism of product chain length determination of type II GGPS identified in the present study may also explain the participation of noncatalytic subunits in the product determination mechanisms... in the model structure of A B Fig Structural information on the product determination mechanism of type II GGPS (A) The direction of the geranylgeranyl chain of GGPP bound in a subunit (blue) of