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J. Biol. Chem., Vol. 281, Issue 21, 14991-15000, May 26, 2006

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Crystal Structure of Type-III Geranylgeranyl Pyrophosphate Synthase from Saccharomyces cerevisiae and the Mechanism of Product Chain Length Determination^*

Tao-Hsin Chang, Rey-Ting Guo, Tzu-Ping Ko^¶, Andrew H.-J. Wang^¶1, and Po-Huang Liang^¶2

From the Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, the Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan, and the ^¶Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

Received for publication, December 2, 2005 , and in revised form, February 24, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate C₂₀ geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether-linked lipid. For short-chain trans-prenyltransferases that synthesize C₁₀-C₂₅ products, bulky amino acid residues generally occupy the fourth or fifth position upstream from the first DDXXD motif to block further elongation of the final products. However, the short-chain type-III GGPPs in eukaryotes lack any large amino acid at these positions. In this study, the first structure of type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98 Å resolution. The structure is composed entirely of 15 -helices joined by connecting loops and is arranged with -helices around a large central cavity. Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. Deletion of the first 9 or 17 amino acids caused the dissociation of dimer into monomer, and the (1-17) mutant showed abolished enzyme activity. In each subunit, an elongated hydrophobic crevice surrounded by D, F, G, H, and I -helices contains two DDXXD motifs at the top for substrate binding with one Mg²⁺ coordinated by Asp⁷⁵, Asp⁷⁹, and four water molecules. It is sealed at the bottom with three large residues of Tyr¹⁰⁷, Phe¹⁰⁸, and His¹³⁹. Compared with the major product C₃₀ synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C₄₀ and C₃₀, respectively, suggesting the most important role of Tyr¹⁰⁷ in determining the product chain length.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Five-carbon isopentenyl pyrophosphate (IPP)³ serves as the building block for the isoprenoid natural products (1). IPP is first converted to its isomer dimethylallyl pyrophosphate (DMAPP) by IPP:DMAPP isomerase (2). Condensation of DAMPP with 1-3 IPP results in C₁₀ geranyl pyrophosphate (GPP), C₁₅ farnesyl pyrophosphate (FPP), and C₂₀ geranylgeranyl pyrophosphate (GGPP) by the corresponding synthases (3). These allylic pyrophosphates, including DMAPP, GPP, FPP, and GGPP, serve as the precursors for hormones, pheromones, visual pigments, photoprotective agents, defensive agents, skeletons of lipid for membranes, and constituents of signal transduction networks (4). From the allylic substrate (FPP in most cases), larger linear polymers can be synthesized through the addition of IPP to the growing chain by a group of prenyltransferases (5). These prenyltransferases either form a cis or trans double bond during each IPP condensation (6). Two conserved aspartate-rich motifs DDXXD, where X encodes any amino acid, found in all trans-prenyltransferases, coordinate with Mg²⁺ for substrate binding and the subsequent reaction (7-10), whereas an Asp in a P-loop of the cis-prenyltransferases is responsible for the similar function (11-13).

In general, trans-prenyltransferases synthesize products up to C₅₀ in chain lengths, which can be further divided into short-chain (C₁₀-C₂₅), medium-chain (C₃₀-C₃₅), and long-chain (C₄₀-C₅₀) (14) and cis-prenyltransferases generate much longer products (15). In the co-crystal structure of FPP synthase (FPPs) with nonreactive thiol analog of the allylic substrate DMAPP and the homoallylic substrate IPP, the first DDXXD binds the pyrophosphate head group of the allylic substrate via Mg²⁺ and the second DDXXD coordinates with two other Mg²⁺ ions to form a trinuclear cluster for pulling the pyrophosphate leaving group of the allylic substrate (16). A bulky amino acid residue at the fourth or fifth position before the first DDXXD motif in helix D (see Fig. 1 for sequence homology) forms a blockage underneath the allylic substrate site to avoid the further elongation of FPP product. Replacement of the large amino acid with a small one removes the blockage and leads to longer products (17-19). For the long-chain C₄₀-octaprenyl pyrophosphate synthase (OPPs), its crystal structure shows a small residue at that position and a large amino acid Phe¹³² from another helix (helix G) seals the bottom of the active site crevice (20). Similar to OPPs, medium-chain C₃₀ hexaprenyl pyrophosphate synthase (HexPPs) also utilizes L164 from helix G for shielding the final product in the middle of the crevice (10).

It is interesting that the short-chain geranylgeranyl pyrophosphate synthases (GGPPs) from different species contain either large or small residues at the fourth or fifth position before the first DDXXD (Fig. 1). By virtue of one IPP condensation reaction with FPP, GGPPs synthesizes C₂₀ GGPP, which serves as a precursor for carotenoids, geranylgeranylated proteins, chlorophylls, and archaeal ether-linked lipid for the cytoplasmic membrane (21). These GGPPs are classified into three types based on their amino acid sequences; type-I GGPPs contains a large amino acid at the fourth or fifth position prior to the DDXXD motif, but type-II (with insertion of two amino acids within the first Asp-rich motif) and type-III GGPPs found in eukaryotes (except plants) have a small amino acid at the corresponding position (see Fig. 1) (22). To rationalize the mechanism of product chain length determination for the type-III GGPPs, we solved the crystal structure of Saccharomyces cerevisiae GGPPs to 1.98 Å resolution as reported here. While bearing a common structural feature with other trans-prenyltransferases, GGPPs structure reveals a unique orientation of its N terminus. The helix A of type-III GGPPs protrudes into the other subunit, which seems to be involved in the tight dimer formation. Mutants truncated by deleting this helix and the following loop region were examined for dimer formation as presented here. On the basis of the structure, we carried out site-directed mutagenesis studies to identify the amino acids critical for product chain length determination. Together with recently solved type-I GGPPs,⁴ our structures enhance the understanding of the chain elongation mechanisms for the short-chain trans-prenyltransferases.

View larger version (85K): [in this window] [in a new window]   FIGURE 1. Alignment of amino acid sequences of S. cerevisiae type-III GGPPs (GGPPs-Sce), Pantoea agglomerans type-II GGPPs (GGPPs-Pag), T. thermophilus type-I GGPPs (GGPPs-Tth; Protein Data Bank accession number 1WMW), the avian FPPs (Protein Data Bank accession number 1FPS), human FPPs (Protein Data Bank accession number 1YQ7 and 1YV5), S. solfataricus HexPPs (HexPPs-Sso, Protein Data Bank accession number 2AZJ), T. maritima OPPs (OPPs-Tma, Protein Data Bank accession number 1V4E), and E. coli OPPs (OPPs-Eco). Identical and similar amino acid residues are shown in black and gray shadow, respectively. The regions of the 15 helices of GGPPs-Sce are indicated at the top. The stars indicate the amino acid residues mutated in this study. The box denotes the fourth and fifth amino acid residues, which are small in long-chain OPPs, medium-chain HexPPs, and type-II and -III GGPPs but large in short-chain FPPs and type-I GGPPs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Purification of GGPPs—The gene encoding GGPPs was cloned from S. cerevisiae genomic DNA by using PCR and the forward primer 5'-ggtattgagggtcgcatggaggccaagatagatg-3' and the reverse primer 5'-agaggagagttagagcctcacaattcggataagtgg-3' into the pET32Xa/LIC vector. In this construct, we included a five-residue sequence (MTKNK) from Thermotoga maritima OPPs between the N terminus of GGPPs and the Factor Xa cleavage sequence to expose the protease site for tag removal. The recombinant GGPPs plasmid was then used to transform Escherichia coli JM109 competent cells that were streaked on a Luria-Bertani (LB) agar plate containing 100 µg/ml ampicillin. Ampicillin-resistant colonies were selected from the agar plate and grown in 5 ml of LB culture containing 100 µg/ml ampicillin overnight at 37 °C. The correct construct by sequencing was subsequently transformed to E. coli BL21 (DE3) for protein expression. The 60-ml overnight culture of a single transformant was used to inoculate 6 liters of fresh LB medium containing 100 µg/ml ampicillin. The cells were grown to A₆₀₀ = 0.6 and induced with 1 mM isopropyl -thiogalactopyranoside at 16 °C. After 16 h, the cells were harvested by centrifugation at 7,000 x g for 15 min to collect the cell paste.

The enzyme purification was conducted at 4 °C. Cell paste was suspended in 75 ml of lysis buffer containing 25 mM Tris-HCl, pH 7.5, and 150 mM NaCl. Cell lysate was prepared with a French pressure cell press (AIM-AMINCO Spectronic Instruments) and centrifuged at 17,000 x g to remove cell debris. The cell-free extract was loaded onto a Ni²⁺-NTA column, which had been previously equilibrated with lysis buffer. The column was washed with 10 mM imidazole followed by 20 mM imidazole-containing buffer. His-tagged GGPPs eluted with 100 mM imidazole was dialyzed twice against 3 liters of buffer (25 mM Tris-HCl, pH 7.5, and 150 mM NaCl) and then subjected to Factor Xa digestion to remove the tag. The mixture was then passed through another Ni²⁺-NTA column, and subsequently untagged GGPPs was eluted with 10 mM imidazole-containing buffer and then dialyzed twice against 3 liters of buffer (25 mM Tris-HCl, pH 7.5, and 150 mM NaCl) for storage. SDS-PAGE analysis was used to check the purity (>95%) of GGPPs and its mutants.

Preparation of Selenomethionine-labeled GGPPs—To produce selenomethionine (SeMet)-labeled GGPPs for x-ray phase solving, the plasmid that GGPPs gene was transformed into a BL21 (DE3)-competent cell. The single transformant was first grown overnight at 37 °C in 50 ml of LB medium containing 100 µg/ml ampicillin. The cells were harvested by centrifugation at 7,000 x g for 15 min. The paste suspended in SeMet minimal medium (24) was then used to inoculate 6 liters of SeMet minimal medium with the supplement of 100 µg/ml seleno-DL-methionine. Isopropyl -thiogalactopyranoside was added to a final concentration of 1 mM for induction after the cell density reached A₆₀₀ = 0.4. The cells were grown for another 96 h at 16 °C and then harvested by centrifugation. The subsequent purification procedure for SeMet GGPPs was the same as for the native GGPPs.

Crystallization and Data Collection—Native and SeMet-labeled GGPPs were crystallized using the hanging drop method from Hampton Research (Laguna Niguel, CA) by mixing 2 µl of the GGPPs solution (10-12 mg/ml in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100) with 2 µl of the mother liquor (0.08 M CH₃COONa, 0.145 M (NH₄)₂SO₄, 13% polyethylene glycol 4000, 7-9% glycerol, and 7-9% 1,2-propanediol) and equilibrating with 500 µl of the mother liquor. Within 7 days at room temperature, crystals grew to dimensions of about 0.4 x 0.15 x 0.15 mm. The x-ray diffraction data for the native GGPPs were collected to 1.98 Å resolution at beam line BL17B2 of the National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan), and the SeMet-labeled GGPPs data were collected to 2.4 Å at 100 K on the Taiwan Contract BL12B2 station at SPring-8 (Hyogo, Japan) by employing the multiple-wavelength anomalous diffraction method. The diffraction data were processed and scaled by using the program HKL2000 (25). These GGPPs crystals belong to the space group P2₁2₁2₁, with typical unit cell parameters of a = 48 Å, b = 116 Å, and c = 129 Å. Each asymmetric unit contained a dimeric GGPPs molecule.

Structure Determination and Refinement—The multiple-wavelength anomalous diffraction data sets in the resolution range of 30 to 2.4 Å were collected at wavelengths of 1.0332 Å (low remote), 0.9799 Å (inflection point), 0.9796 Å (peak), and 0.9537 Å (high remote) at and near the selenium absorption edge. Using SOLVE (26), 20 selenium sites in the asymmetric unit were located, and the phase angles were determined in the single wavelength anomalous diffraction method using the peak wavelength data. Further iterative cycles of density modification, model building, and refinement were carried out with RESOLVE (27), Xtalview (28), and the CNS program (29), respectively. R_free values were calculated by using 5% reflections. The stereochemical quality of the refined structure was checked with the program PROCHECK (30). Each monomer of the refined structure encompassed 322 of the 335 total residues of the GGPPs sequence, with a small disordered region of residues 315-327. 578 well ordered water molecules were also located and included in the model. Refinement statistics are summarized in Table 1. All of the structural diagrams were drawn by using the PyMol software (available on the World Wide Web at pymol.sourceforge.net/) and GRASP (31).

View larger version (73K): [in this window] [in a new window]   FIGURE 2. The structural model of S. cerevisiae GGPPs shown in cylinder and ribbon diagrams. A, N-terminal -helices in the outer layer are shown in blue and purple for the two individual subunits, whereas the six helices in the inner layers are shown in cyan and yellow, respectively. Two small peripheral domains are colored green and orange. The black arrows indicate the location of the active site with the Asp residues in the two DDXXD motifs shown as sticks in cyan and yellow. Two identical subunits are associated into a dimer by forming a four-layer helix bundle (F and G helixes) and a four layer helix arm (A and B helixes). Mg2+ ions observed near the DDXXD motifs are shown as red balls. B, the top view model of S. cerevisiae GGPPs is shown as a ribbon diagram. The color and styles shown here are the same as above. These images were produced using PyMol.

Kinetic Parameters for Mutant GGPPs—For enzyme activity measurements, each mutant GGPPs (0.2 µM Y107A, F108A, H139A, Y107A/F108A, Y107A/H139A, L135A/H139A, F108A/H139A, Y107A/F108A/H139A, or Y107A/F108A//L135A/H139A) was used. The reaction was initiated in 200 µl of solution containing 100 mM Hepes (pH 7.5), various concentrations of FPP and [¹⁴C]IPP as specified below, 50 mM KCl, 0.5 mM MgCl₂, and 0.1% Triton X-100 at 25 °C. The enzyme concentration used in all experiments was determined from its absorbance at 280 nm ([cepsilon] = 20,340 M^-1 cm^-1). The measurements of the kinetic parameters for the wild type and mutants followed our published procedure (32, 33). For IPP K_m determinations, 20 µM FPP was utilized to saturate the enzyme, and IPP concentrations from 0.25 to 400 µM varied with the K_m of IPP for each mutant were employed. For FPP K_m and k_cat measurements, 0.25-32 µM FPP was used along with 50 µM [¹⁴C]IPP, except for the mutants of Y107A/H139A, F108A/H139A, Y107A/F108A/H139A, and Y107A/F108A/L135A/H139A, where 250 µM [¹⁴C]IPP was used. For Y107A/H139A, Y107A/F108A/H139A, and Y107A/F108A/L135A/H139A, the enzyme reaction was initiated by adding 0.1 µM mutants to measure the FPP K_m and k_cat. GPP K_m measurements for wild type and S71Y used 0.25-50 µM GPP along with 50 µM [¹⁴C]IPP. To measure the initial rate, 40-µl portions of the reaction mixture were periodically withdrawn within 10% substrate depletion and then mixed with 10 mM EDTA for reaction termination. The radiolabeled products were then extracted with 1-butanol, and the radioactivities associated with aqueous and butanol phases were separately quantitated by using a Beckman LS6500 scintillation counter. Data of initial rates versus substrate concentrations were analyzed by nonlinear regression of the Michaelis-Menten equation using the KaleidaGraph computer program (Synergy software) to obtain K_m and V_max values. The k_cat was calculated from V_max/[E]. Steady-state activity of the S71Y mutant was assayed under the same condition except that GPP instead of FPP was used.

Product Analysis—The GGPPs reaction containing 1 µM enzyme (wild-type or mutant GGPPs), 10 µM FPP, 150 µM [¹⁴C]IPP, 0.1% Triton X-100, 0.5 mM MgCl₂, and 50 mM KCl in 100 mM Hepes buffer (pH 7.5) was incubated for 6 h at 25 °C. Ten mM EDTA was used to terminate the enzyme reaction. For identification of S71Y product, GPP instead of FPP was used as the allylic substrate. The radiolabeled products were extracted with 1-butanol. The 1-butanol was then evaporated, and the 20% 1-propanol solution containing 4.4 units/ml acidic phosphatase, 0.1% Triton X-100, 50 mM sodium acetate (pH 4.7) was used to convert the products to the corresponding alcohols according to the reported procedure (32). After the pyrophosphate hydrolysis catalyzed by acidic phosphatase was completed, the polyprenols were extracted with n-hexane. The polyprenols were separated on reversed-phase TLC using acetone/water (19:1) as mobile phase. The radiolabeled products were identified by autoradiography using a bioimaging analyzer FUJIFILM BAS-1500 (Japan) according to their R_f (retention factor) values reported (22).

Construction of N-terminal Truncated Mutants—For constructing the genes to express the truncated GGPPs without the first N-terminal 17 amino acids (1-17) or without the first 9 amino acids (1-9), a common reverse primer, 5'-agaggagagttagagcctcacaattcggataagtgg-3', was used with the forward primer 5'-ggtattgagggtcgccaaaatgaaagcttgatttc-3' for (1-17) or 5'-ggtattgagggtcgcaataatgatcctgtttggtc-3' for (1-9). The PCR-amplified products from the template were inserted into the pET32Xa/LIC vector as described above. The deletion was confirmed by sequencing, and correct construct was subsequently transformed to E. coli BL21 (DE3) for protein expression. The truncated proteins were purified using a Ni²⁺-NTA column, and their activities were measured as the other mutant enzymes described above.

Gel Filtration Experiments—The molecular mass of GGPPs was determined on a Superdex 75 10/300 GL High Performance column (1 x 30 cm; Amersham Biosciences) by comparing the elution volume of the GGPPs (1 and 0.2 mg/ml) with those of protein molecular mass standards, including catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa). A buffer of 25 mM Tris-HCl, pH 7.5, and 150 mM NaCl was used to elute the proteins at a flow rate of 0.5 ml/min.

View larger version (59K): [in this window] [in a new window]   FIGURE 3. A, seven homologous proteins, including avian FPPs (in green) with FPP bound, E. coli FPPs (Protein Data Bank accession number IRQI; shown in gray), human FPPs (in blue), T. thermophilus type-I GGPPs (in magenta), S. cerevisiae type-III GGPPs (in cyan) containing one Mg2+ ion (in green), S. solfataricus HexPPs (in orange), and T. maritima OPPs (in purple) are superimposed by the SPDB viewer (23). The FPP and Mg2+ ion observed in the superimposed structures are shown with a ball-and-stick model. According to the structures, the N-terminal 17 residues of S. cerevisiae type-III GGPPs adopt opposite orientation compared with the other prenyltransferases. B, Mg2+ ion (in green) in the active site is octahedrally coordinated with two carboxylate groups from Asp75 and Asp79 of the first DDXXD and four water molecules (in orange). Coordination bonds are shown as yellow dotted lines. The Mg2+ ion binds with the pyrophosphate of FPP and plays a catalytic role according to the previous studies. These images were generated using PyMol.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Active Site—Two conserved DDXXD motifs located on helices D and I, respectively, near the opening of a deep cleft, are proposed to be the substrate-binding pocket (Fig. 2A). In the superimposed three-dimensional structures of this GGPPs with other trans-prenyltransferases (Fig. 3A), this region is coincident with the active site proposed for FPPs. From the potential surface diagram (not shown), this proposed active site pocket is surrounded by the polar (mostly positively charged) residues, including Arg⁸⁴, Arg⁸⁵,His⁶⁸, Lys³⁷, and Arg⁴⁰, which are probably responsible for attracting if not directly interacting with the negatively charged pyrophosphate head groups of the substrates. As revealed by a more focused view of this active site (Fig. 3B), a Mg²⁺ ion is coordinated by Asp⁷⁵ and Asp⁷⁹ from the first DDXXD and four water molecules. The distances of coordination bonds from the Mg²⁺ to Asp⁷⁵, Asp⁷⁹, and four water molecules are 2.06, 2.0, 2.5, 3.08, 3.07, and 2.12 Å, respectively, in subunit and 2.03, 2.06, 2.3, 2.33, 2.8, and 3.04 Å, respectively, in subunit. This Mg²⁺ supposedly coordinates with the pyrophosphate group of FPP. There are two short helices, helix E (Ser⁹⁵-Phe⁹⁹) and helix J (Glu²³¹-Glu²³⁵) on the outer surface of the -cone. The region including the loop between D and E, the loop between I and J, helix J, and the loop between J and K are not part of the active site, but it may switch the top region of the active site between open and closed forms, as shown in FPPs (16) and HexPPs structures (10). The open form may allow the substrates to enter and the final product to release, and the closed form makes the tight binding of the substrates with the enzyme (16).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. A, superimposed structures of S. cerevisiae type-III GGPPs (in cyan) and T. thermophilus type-I GGPPs (in pink). The FPP (in blue) from avian FPPs and the IPP (in orange) from E. coli FPPs shown with a stick model are also superimposed. Trp74 and Ala143 of type-I GGPPs shown in a stick-and-ball model with green color have similar spatial location as Tyr107 and His139 shown in red of type-III GGPPs, respectively. Trp74 at the fifth position before the first DDXXD motif in helix D of type-I GGPPs can be predicted to be a critical residue for chain length determination. B, products synthesized by wild-type and mutant GGPPs. Y107A, F108A, and H139A generated C40 and C30 products, which are longer than the C20 synthesized by the wild type. Tyr107 plays a more important role in chain length determination. S71Y synthesizes shorter product C15, a single condensation between the bound GPP and IPP. Their product distributions are summarized in Table 3. C, the molecular ruler mechanism for chain length determination catalyzed by the type-III GGPPs. The final product of C20 GGPP shown in black is shielded by Tyr107 and His139 at the bottom. The H139A mutation has yielded smaller space for product elongation through helix G (in purple). However, Y107A provides more space to accumulate longer product (C40) along helix F (in green). The substitution of Ser71, the fourth amino acid prior to the first DDXXD motif, with a large residue of Tyr caused the formation of C15.

Reaction Kinetics and Final Products of Different Mutants—To test the above hypothesis, we replaced these large residues Tyr¹⁰⁷, Phe¹⁰⁸, and His¹³⁹ with Ala and examined the chain lengths of their products. Less than 5-fold changes of enzyme k_cat values for the mutants as compared with the wild type (see Table 2) were observed. However, the FPP K_m of Y107A/F108A/H139A was decreased 9-fold, indicating that this triple mutant has higher FPP affinity. None of these single mutations or the mutations on other sites has caused significantly reduced FPP K_m. The increase of IPP K_m values by 120, 65, 22, 340, and 170-fold compared with the wild type were observed in the mutants of Y107A/F108A/H139A, Y107A/F108A/L135A/H139A, L135A/H139A, Y107A/H139A, and F108A/H139A, respectively, indicating significantly lower affinity of these mutants with IPP. However, under the high concentrations of FPP (10 µM) and IPP (150 µM), which were used to yield the products by all of the mutants as shown below, the activities were not significantly changed (less than 5-fold). Further increase of the substrate concentrations (up to 20 µM FPP and 320 µM [¹⁴C]IPP) did not lead to different final products (data not shown).

Compared with the previously identified His¹³⁹ from mutagenesis study (22), Tyr¹⁰⁷ plays a more dominant role in determining product chain length. According to our crystal structure, the side chains of Tyr¹⁰⁷ and His¹³⁹ point into the tunnel interior from opposite -helixes (Fig. 4A, top, for the side view). From the top view (bottom of Fig. 4A), Tyr¹⁰⁷ and His¹³⁹ are well positioned to act as the "double floor" in the hollow tunnel, with Tyr¹⁰⁷ occupying the major part.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. Size column elution profile for determining the molecular species of wild type (dotted and dashed line), (1-9) (solid line), and (1-17) GGPPs (dotted line). The wild type (1 and 0.2 mg/ml) was eluted as a dimer, but (1-9) and (1-17) GGPPs (1 mg/ml) were both eluted later, corresponding to a monomer. Only the wild type contained an extra five amino acids in the N terminus for tag cleavage (see "Experimental Procedures"). mAU, 10-3 x absorbance unit.

View larger version (48K): [in this window] [in a new window]   FIGURE 6. A, the cylindrical diagram showing the essential amino acids for product chain length determination from short-, medium-, and long-chain trans-prenyltransferases. The common active site crevice surrounded by helices D, F, and G with the bulky amino acids at the bottom for different trans-prenyltransferases are displayed. The key residues for chain length determination are Phe113 for C15-avian FPPs, Trp74 for C20-T. thermophilus type-I GGPPs, Tyr107 and His139 for C20-S. cerevisiae type-III GGPPs, Leu164 for C30-S. solfataricus HexPPs, and Phe132 for C40-T. maritima OPPs, which are shown in green, magenta, cyan, brown, and purple, respectively. B, the active site cavity of avian FPPs, T. thermophilus type-I GGPPs, S. cerevisiae type-III GGPPs, S. solfataricus HexPPs, and T. maritima OPPs shown in green, magenta, cyan, orange, and purple, respectively. Right,ina ribbon diagram, the DDXXD motifs and the key residues for chain length determination are colored with blue and gray and red and green in a ball-and-stick model, respectively. The distances between the first Asp of the first DDXXD motif and Phe113, Trp74, Tyr107/His139, Leu164, and Phe132 are 6.77, 9.77, 8.86/10.85, 12.3, and 19.07 Å for FPPs, type-I GGPPs, type-III GGPPs, HexPPs, and OPPs, respectively. Left, electrostatic surface potential diagrams are color-coded from red to blue according to charge potential from -15 to 15 kBT (where kB Boltzmann constant and T is temperature in Kelvin). The scope of the green dashed circles is restricted from the first Asp of the first DDXXD motif at the top to the bulky resides for chain length determination at the bottom. The active site tunnels of these trans-prenyltransferases are deeper and wider with the increased chain lengths of the ultimate products. These diagrams were generated with PyMol and GRASP (31).

The Function of N-terminal Helix A in Dimer Formation—As shown in Fig. 3A, an unexpected feature in the crystal structure of type-III GGPPs is the orientation of the first N-terminal 17 amino acids, which protrude into and hold the other subunit. In order to elucidate the possible function of the N-terminal residues in dimer formation, the truncated (1-9) and (1-17) GGPPs, by deleting the first 9 amino acids (helix A) and 17 amino acids (helix A plus a loop), were constructed. According to size exclusion chromatography (Fig. 5), both of the truncated proteins were a monomer at 1 mg/ml, whereas the wild-type GGPPs formed a dimer at 1 mg/ml and lower protein concentration (0.2 mg/ml). (1-9) showed a 325-fold lower activity compared with the wild type, but (1-17) had no detectable activity. The CD spectra of wild type, (1-9), and (1-17) were similar (data not shown), indicating the deletion did not change the secondary structures. A subtle change in active site conformation must occur to account for the activity reduction for the truncated mutants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we solved the three-dimensional structure of type-III GGPPs from S. cerevisiae. This enzyme shares mostly the same folding as the available structures of avian FPPs (8), Thermus thermophilus type-I GGPPs,⁴ S. solfataricus HexPPs (10), and T. maritima OPPs (9), although it shares only 22, 23, 22, and 25% sequence identity and 39, 40, 45, and 45% similarity, respectively, with those prenyltransferases. Compared with the type-I GGPPs, the superposition is better in helices D, E, F, G, H, I, K, and N with root mean square deviation = 1.53 Å for the fitting of 648 C atoms of the dimeric type-III GGPPs. The most different feature in the crystal structures of type-III GGPPs is the location of the N-terminal helix A, which embraces the other subunit. Deletion of the first nine amino acids (the helical region) is sufficient to dissociate the dimer into monomer, supporting the importance of the N-terminal helix (helix A) in dimer formation. For other trans-prenyltransferases, however, their N-terminal helix is located within its own subunit, and the dimer is formed mainly through the interfacial interaction from helices E and F (e.g. OPPs and HexPPs) without the contribution from this N-terminal helix. Our data also indicate the monomer is remarkably less active, although each monomer contains a separate active site. To our knowledge, this is the first case in which a dimeric prenyltransferase can be disrupted into a monomer by a mutation (a deletion here). The structure of the monomer will be further studied.

It was mysterious that unlike the other short-chain trans-prenyltransferases, the fourth and the fifth amino acids prior to the first DDXXD motif (the FPP binding site) of type-III GGPPs are not large amino acids. On the basis of crystal structure, we have replaced some large amino acids at the bottom of the active site crevice to test their role in chain length determination. Some mutants show different kinetic parameters (smaller FPP K_m and larger IPP K_m values) compared with the wild type. This was also observed for the mutants of FPPs that became GGPPs by random mutagenesis (18). The calculation of kinetic parameters followed our previous reports on the kinetics of prenyltransferases (32, 33). Although the prenyltransferases and their mutants produced various products, all of the products can be extracted with 1-butanol. We also demonstrated that each IPP condensation step has a similar rate constant (32, 33), so the k_cat could represent the IPP condensation rate constant. Here the k_cat values of the GGPP mutants, which made multiple products, were calculated from the initial rates of the substrate depletion and should represent the steady-state activity for forming their favorite elongated products due to the removal of the blockage by replacing the large amino acids at the bottom of active crevice with a small Ala. The final products of the mutants described here were also generated under sufficient quantities of FPP and [¹⁴C]IPP.

From the structure and mutagenesis analysis presented above, we discovered an important residue, Tyr¹⁰⁷, in addition to the previously identified His¹³⁹ (22), which seals the bottom of the active site crevice to control the product chain length. The substitution of the large Tyr¹⁰⁷ and His¹³⁹ with a smaller Ala removes the "floor" of the crevice, thereby allowing for the formation of longer chain length products. This kind of "floor" has been found in other trans-prenyltransferases and also the protein prenyltransferases, which catalyze the transfer of farnesyl or geranylgeranyl group into the Cys of the CAAX motif (where A is an aliphatic amino acid) of the protein substrates, indicating a common mechanism of molecular ruler (34). However, distinct from other prenyltransferases, the "floor" in type-III GGPPs is a "double floor" constituted of two large amino acids. As illustrated in Fig. 6A, the "floor" is located at helix D for FPPs and type-I GGPPs but in helix F and G for type-III GGPPs and in helix G for OPPs and HexPPs. The key residues for chain length determination are Phe¹¹³ for C₁₅-avian FPPs, Trp⁷⁴ for C₂₀-T. thermophilus type-I GGPPs, Tyr¹⁰⁷ and His¹³⁹ for C₂₀-S. cerevisiae type-III GGPPs, Leu¹⁶⁴ for C₃₀-S. solfataricus HexPPs, and Phe¹³² for C₄₀-T. maritima OPPs (Fig. 6A). According to these known three-dimensional structures, the distances between the first Asp of the first DDXXD motif and Phe¹¹³, Trp⁷⁴, Tyr¹⁰⁷/His¹³⁹, Leu¹⁶⁴, and Phe¹³² for FPPs, Type-I GGPPs, Type-III GGPPs, HexPPs, and OPPs, respectively, are increasing with the longer product chain lengths (Fig. 6B, left). The areas inside the circled cavity of these prenyltransferases are also larger with the increasing size of the ultimate products catalyzed by these enzymes (Fig. 6B, right).

Type-I GGPPs utilizes Trp⁷⁴ at helix D, and type-III GGPPs uses Tyr¹⁰⁷ and His¹³⁹ at different helices, F and G, to control the product chain length although both enzymes make the same product. Based on the different strategies in sealing the active site, our data suggest that type-I GGPPs may evolve from FPPs by shifting the large amino acid from the fourth to fifth position before the first DDXXD to shield the product, whereas type-III GGPPs and medium-chain and long-chain prenyltransferases may evolve from the common ancestor. Together, the data significantly extend our understanding on the biosynthesis of short-chain polyprenyl molecules.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2DH4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Science Council Grant NSC94-2311-B-001-034 and a grant from Academia Sinica (to P.-H. L.). The Biological Crystallography Facilities (Taiwan Beamline BL12B2 at SPring-8) were supported by the National Science Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Institute of Biological Chemistry, Academia Sinica, 128 Academia Rd., Taipei 115, Taiwan. Tel.: 886-2-2785-5696 (ext. 5010); Fax: 886-2-2788-9759; E-mail: ahjwang{at}gate.sinica.edu.tw. ² To whom correspondence may be addressed: Institute of Biological Chemistry, Academia Sinica, 128 Academia Rd., Taipei 115, Taiwan. Tel.: 886-2-2785-5696 (ext. 6070); Fax: 886-2-2788-9759; E-mail: phliang{at}gate.sinica.edu.tw.

³ The abbreviations used are: IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; FPPs, farnesyl pyrophosphate synthase; OPPs, octaprenyl pyrophosphate synthase; GGPPs, geranylgeranyl pyrophosphate synthase; SeMet, selenomethionine; NTA, nitrilotriacetic acid; HexPPs, hexaprenyl pyrophosphate synthase.

⁴ Crystal structure of geranylgeranyl diphosphate synthase from T. thermophilus, deposited by K. Suto, K. Nishio, Y. Nodake, K. Hamada, M. Kawamoto, N. Nakagawa, S. Kuramitu, and K. Miura, Riken Structural Genomics Proteomics and Initiative (Rsgi) on July 21, 2004.

	ACKNOWLEDGMENTS

 
The synchrotron data collection was conducted at the Biological Crystallography Facilities (Taiwan Beamline BL12B2 at SPring-8).

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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