Geranylgeranyl Diphosphate Synthase Activity Research Articles

Enhancing the Catalytic Activity of Geranylgeranyl Diphosphate Synthase through Ancestral Sequence Reconstruction and Semirational Design.

Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.

A Simple In Vitro Assay to Measure the Activity of Geranylgeranyl Diphosphate Synthase and Other Short-Chain Prenyltransferases.

Most carotenoids are C40 metabolites produced from C20 geranylgeranyl diphosphate (GGPP). The enzymes that produce this precursor, GGPP synthases (GGPPS), are members of the short-chain prenyltransferase (SC-PT) family. SC-PTs are enzymes that catalyze the sequential head-to-tail addition of one or more C5 molecules of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) with the concomitant release of pyrophosphate (PPi). SC-PTs produce linear isoprenyl diphosphates of up to C20 (GGPP) that serve as precursors for many groups of isoprenoids with a wide range of essential biological functions in Eucarya, Bacteria, and Archaea. Enzymatic analysis of SC-PT activity normally requires complex, laborious and expensive methods such as radioactivity-based assays or liquid chromatography-mass spectrometry (LC-MS). Here we describe a fast and inexpensive spectrophotometric protocol for determining the kinetic parameters of SC-PTs in purified enzyme preparations, using an adapted assay for PPi quantification. We developed the method using the Arabidopsis thaliana GGPPS11 enzyme, which produces geranylgeranyl diphosphate for the synthesis of carotenoids in the chloroplast.

Reduced Activity of Geranylgeranyl Diphosphate Synthase Mutant Is Involved in Bisphosphonate-Induced Atypical Fractures

Bisphosphonates are widely used for treating osteoporosis, a common disorder in which bone strength is reduced, increasing the risk for fractures. Rarely, bisphosphonates can paradoxically lead to atypical fractures occurring spontaneously or with trivial trauma. Recently, a novel missense mutation (D188Y) in the GGPS1 gene, encoding for geranylgeranyl diphosphate synthase (GGPPS), was associated with bisphosphonate-induced atypical fractures. However, the molecular basis for GGPPS involvement in this devastating condition remains elusive. Here, we show that while maintaining an overall unperturbed global enzyme structure, the D188Y mutation leads to ∼4-fold catalytic activity decrease. Furthermore, GGPPS-D188Y is unable to support cross-species complementation, highlighting the functional significance of the reduced catalytic activity observed in vitro. We next determined the crystal structure of apo-GGPPS-D188Y, revealing that while Y188 does not alter the protein fold, its bulky side chain sterically interferes with substrate binding. In agreement, we show that GGPPS-D188Y exhibits ∼3-fold reduction in the binding affinity of zoledronate, a commonly used bisphosphonate. However, inhibition of the mutated enzyme by zoledronate, in pharmacologically relevant concentrations, is maintained. Finally, we determined the crystal structure of zoledronate-bound GGPPS-D188Y, revealing large ligand-induced binding pocket rearrangements, revising the previous model for GGPPS-bisphosphonate interactions. In conclusion, we propose that among heterozygotes residual GGPPS activity is sufficient to support physiologic cellular function, concealing any pathologic phenotype. However, under bisphosphonate treatment, GGPPS activity is reduced below a crucial threshold for osteoclast function, leading to impaired bone remodeling and increased susceptibility to atypical fractures.

Control Activity of Yeast Geranylgeranyl Diphosphate Synthase from Dimer Interface through H-Bonds and Hydrophobic Interaction

Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased k(cat) values (200-250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS.

Combined Experimental and Theoretical Study of Long-Range Interactions Modulating Dimerization and Activity of Yeast Geranylgeranyl Diphosphate Synthase

We present here how two amino acid residues in the first helix distal from the main dimer interface modulate the dimerization and activity of a geranylgeranyl diphosphate synthase (GGPPs). The enzyme catalyzes condensation of farnesyl diphosphate and isopentenyl diphosphate to generate a C(20) product as a precursor for chlorophylls, carotenoids, and geranylgeranylated proteins. The 3D structure of GGPPs from Saccharomyces cerevisiae reveals an unique positioning of the N-terminal helix A, which protrudes into the other subunit and stabilizes dimerization, although it is far from the main dimer interface. Through a series of mutants that were characterized by analytic ultracentrifugation (AUC), the replacement of L8 and I9 at this helix with Gly was found sufficient to disrupt the dimer into a monomer, leading to at least 10(3)-fold reduction in activity. Molecular dynamics simulations and free energy decomposition analyses revealed the possible effects of the mutations on the protein structures and several critical interactions for maintaining dimerization. Further site-directed mutagenesis and AUC studies elucidated the molecular mechanism for modulating dimerization and activity by long-range interactions.

Cloning and characterization of isoprenyl diphosphate synthases with farnesyl diphosphate and geranylgeranyl diphosphate synthase activity from Norway spruce ( Picea abies) and their relation to induced oleoresin formation

The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene ( PaIDS 4) encodes a farnesyl diphosphate synthase and the other two ( PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.

Geranylgeranyl Diphosphate Synthase in Fission Yeast Is a Heteromer of Farnesyl Diphosphate Synthase (FPS), Fps1, and an FPS-like Protein, Spo9, Essential for Sporulation

Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

Isoprenoids are the most diverse and abundant group of natural products. In plants, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors to many isoprenoids having essential functions. Terpenoids and sterols are derived from FPP, whereas gibberellins, carotenoids, casbenes, taxenes, and others originate from GGPP. The corresponding synthases (FPP synthase [FPPS] and GGPP synthase [GGPPS]) catalyze, respectively, the addition of two and three isopentenyl diphosphate molecules to dimethylallyl diphosphate. Maize (Zea mays L. cv B73) endosperm cDNAs encoding isoprenoid synthases were isolated by functional complementation of Escherichia coli cells carrying a bacterial gene cluster encoding all pathway enzymes needed for carotenoid biosynthesis, except for GGPPS. This approach indicated that the maize gene products were functional GGPPS enzymes. Yet, the predicted enzyme sequences revealed FPPS motifs and homology with FPPS enzymes. In vitro assays demonstrated that indeed these maize enzymes produced both FPP and GGPP and that the N-terminal sequence affected the ratio of FPP to GGPP. Their functionality in E. coli demonstrated that these maize enzymes can be coupled with a metabolon to provide isoprenoid substrates for pathway use, and suggests that enzyme bifunctionality can be harnessed. The maize cDNAs are encoded by a small gene family whose transcripts are prevalent in endosperm beginning mid development. These maize cDNAs will be valuable tools for assessing the critical structural properties determining prenyl transferase specificity and in metabolic engineering of isoprenoid pathways, especially in cereal crops.

An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in taxol biosynthesis, we cloned, characterized and functionally expressed the GGPP synthase gene from Taxus media. A 3743-bp genomic sequence of T. media was isolated by genome walking strategy which contained an 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed high similarity to other plant GGPPSs. Subsequently the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene and its deduced polypeptide contained all the five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, angiosperm and gymnosperm classes, which might have evolved in parallel from the same ancestor. To our knowledge this was the first report that the geranylgeranyl diphosphate synthase genes were free of intron and evolved in parallel between angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed in yeast mutant (SFNY368) lacking of GGPP synthase activity through functional complementation, and the transgenic yeast showed to have activity of GGPP synthase. This was also the first time to use SFNY368 to identify the function of plant-derived GGPPSs. Furthermore, investigation of the impact of methyl jasmonate (MeJA) on the expression of TmGGPPS revealed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.

Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give ( all- E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K m values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni 2+-affinity column were 24.1, 6.8, 2.3, and 11.5 μM, respectively. The enzyme shows optimum activity at approximately 40 °C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.

Improvement of cephalomanine production in Taxus chinensis cells by a combination of sucrose and methyl jasmonate

Cell suspension cultures of Taxus chinensis, supplemented with 25 g sucrose l−1, produced 11 mg cephalomanine l−1, 21 g biomass l−1 and 19 nkat geranylgeranyl diphosphate (GGPP) synthase activity g protein−1. Supplementation of the cultures with 100 μM methyl jasmonate (MJA) produced 17 mg cephalomanine l−1, 6 g biomass l−1 and 78 nkat GGPP synthase activity g protein−1. Addition of sucrose and MJA together produced 24 mg cephalomanine l−1, 18 g biomass l−1 and 55 nkat GGPP synthase activity g protein−1.

Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase.

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.

Induction of geranylgeranyl diphosphate synthase activity and taxane accumulation in Taxus baccata cell cultures after elicitation by methyl jasmonate

The results presented in this paper describe for the first time the presence of an inducible enzyme in the biosynthetic pathway leading to the anticancer drug paclitaxel. Methyl jasmonate, when administered to Taxus baccata cell suspension cultures, was found to rapidly induce geranylgeranyl diphosphate (GGPP) synthase activity. Experiments showed that after elicitation at day 7 of the subculturing period, the enzyme activity is rapidly induced during the first 6 h and reaches its maximum of 60.7 μkat/kg of protein after 5 days. The enzyme’s induction was followed by a sharp increase in taxane accumulation in the cells, which started 24 h after elicitation and reached its maximum after 5 days. In addition taxine-B was found in the medium of unelicited cultures but not in the medium of elicited cells.

Geranylgeranyl diphosphate synthase activity and taxane production in Taxus baccata cells

A Taxus baccata cell line has been characterized for growth, nutrient uptake, taxane production and for GGPP synthase activity. The enzyme is extracted by inclusion of non-ionic detergent and its activity parallels taxane production for the first three days. The enzyme activity reaches its peak at day 3 of culture growth, while total taxane formation rises until day 9, when the maximum taxane accumulation in the cells is observed (∼0.004% dry wt). After this day the taxane excretion in the medium increases, reaching its maximum at day 19.

Cloning and Functional Expression of a cDNA Encoding Geranylgeranyl Diphosphate Synthase fromTaxus canadensisand Assessment of the Role of this Prenyltransferase in Cells Induced for Taxol Production

Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-inducedTaxus canadensissuspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an inducedT. canadensiscell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90–100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative “mature proteins” from which the transit peptide was deleted, were transformed into a yeast mutant defective for the β-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction.In vitroactivity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS–PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in inducedT. canadensissuspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.

Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping and tissue expression

We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitiously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.—Ericsson, J., J. M. Greene, K. C. Carter, B. K. Shell, D. R. Duan, C. Florence, and P. A. Edwards. Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping, and tissue expression.

Conversion from Farnesyl Diphosphate Synthase to Geranylgeranyl Diphosphate Synthase by Random Chemical Mutagenesis

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.

On the use of the name "Catskill."

Bisphosphonates are widely used for treating osteoporosis, a common disorder in which bone strength is reduced, increasing the risk for fractures. Rarely, bisphosphonates can paradoxically lead to atypical fractures occurring spontaneously or with trivial trauma. Recently, a novel missense mutation (D188Y) in the <i>GGPS1</i> gene, encoding for geranylgeranyl diphosphate synthase (GGPPS), was associated with bisphosphonate-induced atypical fractures. However, the molecular basis for GGPPS involvement in this devastating condition remains elusive. Here, we show that while maintaining an overall unperturbed global enzyme structure, the D188Y mutation leads to ∼4-fold catalytic activity decrease. Furthermore, GGPPS-D188Y is unable to support cross-species complementation, highlighting the functional significance of the reduced catalytic activity observed in vitro. We next determined the crystal structure of apo-GGPPS-D188Y, revealing that while Y188 does not alter the protein fold, its bulky side chain sterically interferes with substrate binding. In agreement, we show that GGPPS-D188Y exhibits ∼3-fold reduction in the binding affinity of zoledronate, a commonly used bisphosphonate. However, inhibition of the mutated enzyme by zoledronate, in pharmacologically relevant concentrations, is maintained. Finally, we determined the crystal structure of zoledronate-bound GGPPS-D188Y, revealing large ligand-induced binding pocket rearrangements, revising the previous model for GGPPS-bisphosphonate interactions. In conclusion, we propose that among heterozygotes residual GGPPS activity is sufficient to support physiologic cellular function, concealing any pathologic phenotype. However, under bisphosphonate treatment, GGPPS activity is reduced below a crucial threshold for osteoclast function, leading to impaired bone remodeling and increased susceptibility to atypical fractures.