©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Conversion from Farnesyl Diphosphate Synthase to Geranylgeranyl Diphosphate Synthase by Random Chemical Mutagenesis (*)

(Received for publication, December 1, 1995; and in revised form, January 18, 1996)

Shin-ichi Ohnuma (1) (2) Takeshi Nakazawa (1) Hisashi Hemmi (1) Anna-Maria Hallberg (1) Tanetoshi Koyama (1) Kyozo Ogura Tokuzo Nishino (1) (2)

From the  (1)Department of Biochemistry and Engineering, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-77 Japan and the (2)Institute for Chemical Reaction Science, Tohoku University, Sendai 980, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 NaNO(2) 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 12 Å 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 Kfor dimethylallyl diphosphate, decrease of V(max) for dimethylallyl diphosphate, and allylic substrate dependence of K for IPP.


INTRODUCTION

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) (^1)with allylic diphosphates to synthesize prenyl diphosphates with various chain lengths. These enzymes are classified according to the chain length of the final product and the geometry of the double bond that is formed by the condensation. So far a number of prenyltransferases have been found from various organisms and characterized(1) . For example, FPP synthase, which is a key enzyme of the biosynthesis of steroids, prenyl quinones, farnesylated protein, and dolichols, catalyzes the condensations of IPP with DMAPP (C(5)) and with geranyl diphosphate (GPP, C) to give FPP (C) as the ultimate product (Fig. 1A). GGPP synthase, whose product is a precursor of carotenoids, geranylgeranylated proteins, and ether-linked lipids of archaebacterium, utilizes DMAPP, GPP, and FPP as allylic substrates to give an amphiphilic molecule containing 4 isoprene units, GGPP (C). Solanesyl diphosphate synthase catalyzes the consecutive condensation of IPP with E-stereochemistry to produce a C compound. Although these enzymes catalyze similar condensation reactions, they do not catalyze the condensation beyond the limit of the chain length of product determined by their own specificities. Why does the condensation stop at the step that is determined by each enzyme?


Figure 1: Pathway of isoprenoid biosynthesis (A) and schematic diagram of the red-white screening (B). A, wild-type FPP synthase of B. stearothermophilus catalyzes consecutive condensations of IPP to produce FPP as an ultimate product. We selected mutated FPP synthase that has GGPP synthase activity by using the color selection system, which utilizes carotenoid synthetic genes (crtB and crtI). B, E. coli DH5alpha was transformed with pACYC-IB, which expresses phytoene synthase (crtB) and phytoene desaturase (crtI). The cells were transformed with the plasmids derived from the library of random mutated FPP synthase, plated on LB plate, and then red colonies, which mean that the mutated FPP synthases have GGPP synthase activity, were isolated.



The prenyl chain length of respiratory quinones is altered by viral infection and differs from tissue to tissue. The dolichyl chain length in rat liver also changes on carcinogenesis (2) or aging(3, 4) . It has also been reported that the product chain length of prenyltransferases is changed under some reaction conditions(5, 6, 7, 8) . In all cases, the chain length always changes shorter than the ultimate chain length. Ohnuma et al.(9) and Matsuoka et al.(10) have suggested on the basis of in vitro examinations that these phenomena reflect the level of IPP and metal ions in the living cells. However, from these lines of evidence, it is difficult to understand the mechanisms that force each prenyltransferase to yield its intrinsic product.

During the past few years the amino acid sequences of FPP synthases (11, 12, 13, 14, 15) , GGPP synthases(16, 17, 18, 19, 20) , hexaprenyl diphosphate synthase (21) , heptaprenyl diphosphate synthase(22) , and octaprenyl diphosphate synthase (23) have been determined. Comparisons of the primary structures revealed several conserved domains including two aspartate-rich domains, DDXXD, where X encodes any amino acid. In FPP synthase, site-directed mutagenesis studies have been carried out by several groups (23, 24, 25, 26, 27) with special attention to the two aspartate-rich domains. These studies have indicated that the aspartate-rich domains are essential for catalytic activity. It is suggested that the aspartate residues bind the diphosphate moieties of IPP and allylic substrate through a magnesium bridge. However, none of the previous studies answered the question which amino acid residues are important in determining the chain length of the ultimate product.

In order to obtain information about amino acid residues that are related to chain-length determination, we tried to convert FPP synthase to GGPP synthase using random chemical mutagenesis. If combined with biological selection, the random mutagenesis provides a powerful method for identifying important amino acid residues. Recently, we have developed an in vivo method for detecting GGPP synthase activity, which utilizes carotenoid biosynthesis genes of Erwinia uredovora to visualize a colored clone expressing GGPP synthase activity(19) . We introduced random mutations on the Bacillus stearothermophilus FPP synthase gene using NaNO(2), and screened the clones that showed GGPP synthase activity by taking advantage of the color selection method (Fig. 1). This paper reports the determination of the amino acid residues that are important for chain length determination.


EXPERIMENTAL PROCEDURES

Materials

Precoated reversed phase thin layer chromatography plates, LKC-18 were purchased from Whatman Chemical Separation, Inc. Precoated normal phase thin layer chromatography plates, Kieselgel 60 were purchased from E. Merck. (all-E)-FPP, (all-E)-GGPP, GPP, and DMAPP were the same preparations as used in the previous work(19) . [1-^14C]IPP was purchased from Amersham. Avian myeloblastosis virus reverse transcriptase was obtained from Life Science, Inc. pTrc99A and pTV118N were purchased from Pharmacia Biotech and Takara Shuzo Co., Ltd, respectively. All other chemicals were of analytical grade.

Random Mutagenesis of FPP Synthase Gene

Introduction of random mutations in the structural gene for the FPP synthase was carried out using nitrous acid as described by Myers et al. (28) with some modifications. To obtain single-stranded DNA templates, the NcoI/HindIII fragment from pEX11(14) , which contained the B. stearothermophilus wild-type FPP synthase gene, was subcloned into pTV118N (Takara), yielding pFPS. Then single-stranded DNA was isolated with a helper phage, M13KO7. The single-stranded DNA was treated with 0.25 or 1 M of sodium nitrite for 30 min, and then the mutagenized plasmids were purified. The double-stranded DNA was synthesized by the action of avian myeloblastosis virus reverse transcriptase using DNA primer, RV-N (Takara). The fragments of the FPP synthase gene were excised from the plasmids with NcoI and HindIII, ligated with NcoI and HindIII cut dephosphorylated pTV118N, and transformed into Escherichia coli DH5alpha.

Screening of the Mutants Expressing GGPP Synthase Activity

E. coli DH5alpha was transformed with plasmid pACYC-IB, which contained crtB and crtI genes (19) , and the competent cells that contained pACYC-IB were made. The competent cells were transformed with the plasmids that were isolated from the two libraries derived from the treatments of 0.25 and 1 M NaNO(2) and spread on the LB plate containing tetracycline (50 µg/ml) and ampicillin (50 µg/ml). Since the competent cells express phytoene synthase (crtB) and phytoene desaturase (crtI), those transformed by a plasmid that encodes mutant FPP synthase showing GGPP synthase activity should produce lycopene and become red. One red positive clone was obtained from about 16,700 transformants of the former library. On the other hand, 10 red positive clones were obtained from about 7,600 transformants of the latter library. Four plasmids (pMU1-4) were isolated from the red-colored cells, and each clone was analyzed. The plasmids, pMU1Trc, pMU2Trc, pMU3Trc, and pMU4Trc were also constructed by introduction of the NcoI-HindIII fragment of pMU1, pMU2, pMU3, and pMU4, into the multicloning site of pTrc99A, respectively, in order to compare the activities with that reported previously(27) . The DNA sequences of all mutants were determined.

Construction of the Mutated FPP Synthase by Fragment Replacement and Site-directed Mutagenesis

To determine the essential amino acid residues in the mutagenized FPP synthase showing GGPP synthase activity, we first constructed several plasmids by replacing fragments. pMU1A and pMU1C were constructed from pEX11 by replacing the NcoI-EagI and NruI-HindIII fragments with the corresponding fragments of pMU1, respectively. Replacing the NcoI-SnaBI, EagI-NruI, and NruI-HindIII fragments of pEX11 with the corresponding fragments of pMU3 resulted in pMU3A, pMU3B, and pMU3C, respectively. Replacement of the NcoI-EagI and NruI-HindIII fragments of pEX11 with the corresponding fragments of pMU4 resulted in pMU4A and pMU4C, respectively. Site-directed mutagenesis was performed by the Kunkel mutagenesis(29) . Single strand wild-type FPP synthase gene, used as a template in the mutagenesis reaction, was generated by M13KO7 helper phage infection of XL1-Blue MRF` (Stratagene) cells that contained pFPS. The resulting antisense single strand DNA template was isolated, purified by standard methods, and sequenced to confirm identity. The synthetic sense oligonucleotides designed to produce the desired point mutations were as follows: Y81H, 5`-GATCCATACGCACTCTTTG-3`; L34V, 5`-GAAGGGCCGGCGAAGGTGAAAAAGG-3`; R59Q, 5`-CCACCGTTCAGGCGCTCGGCAAAG-3`; V157A, 5`-GGGATGGCCGCCGGTCAGGCAGC-3`; H182Y, 5`-CATTCATCGGTATAAAACCGGG-3`. After mutagenesis, all mutants were confirmed by DNA sequencing. The E. coli DH5alpha containing pACYC-IB was transformed with the plasmids carrying mutated FPP synthase genes, and then the color of the colonies was checked.

Preparation and Purification of Mutated FPP Synthase

E. coli DH5alpha was transformed with each of the plasmids, pMU1, pMU2, pMU3, pMU4, and cultured according to the methods described previously(30) . The cells were harvested and disrupted by sonication in 50 mM Tris-HCl buffer, pH 7.0, containing 10 mM 2-mercaptoethanol and 1 mM EDTA. The homogenate was heated at 55 °C for 60 min and then centrifuged at 100,000 times g for 10 min. The supernatant was used as a crude enzyme to assay for prenyltransferase activity. Further purification of the mutated enzymes was carried out essentially according to the methods described previously(14) . Each of the mutated FPP synthases was confirmed to be homogeneous by SDS-polyacrylamide gel electrophoresis (12.5%) with Coomassie Brilliant Blue staining. All the mutated FPP synthase, mutant 1, 2, 3, 4, showed similar chromatographic properties to those of wild-type enzyme during the purification procedures, but the amount of proteins produced were different (Table 1).



Measurement of Prenyltransferase Activity

The enzyme activity was measured by determination of the amount of [1-^14C]IPP incorporated into butanol-extractable polyprenyl diphosphate according to the method described previously (14) . One unit of enzyme activity was defined as the activity required to incorporate 1 nmol of [1-^14C]IPP into products when the assay was carried out using GPP as an allylic substrate.

Product Analysis

After enzymatic reaction at 55 °C, the polyprenyl diphosphates were extracted with 1-butanol, and then the 1-butanol was evaporated under N(2) stream. The resulting polyprenyl diphosphates were treated with acid phosphatase according to the method of Fujii et al.(31) . The hydrolysates were extracted with pentane and analyzed by reversed phase thin layer chromatography using LKC-18 developed with acetone/H(2)O (9/1) and normal phase thin layer chromatography using Kieselgel 60 developed with benzene/ethyl acetate (9/1). Authentic standard alcohols were visualized with iodine vapor, and the distribution of radioactivity was detected with a Bio-image analyzer BAS2000 (FUJIFILM).


RESULTS

Random Mutagenesis of FPP Synthase Gene and Isolation of Mutants Showing GGPP Synthase Activity

A random chemical mutagenesis strategy using a single-stranded DNA was used to introduce mutations in the entire FPP synthase gene from B. stearothermophilus. Single-stranded DNA derived from pFPS was subjected to chemical treatment with 0.25 or 1 M NaNO(2) for 30 min, and then two plasmid libraries containing mutated FPP synthase genes were constructed. Conditions employed for these mutagenizing were weaker than those described by Myers et al.(28) .

In order to screen the mutants having GGPP synthase activity, the red-white screening system reported previously (19) was used (Fig. 1). In this system, if the GGPP synthase activity is expressed in the transformant E. coli cell that expresses the genes for phytoene synthase and phytoene desaturase, the transformant should produce lycopene and become red. Before screening, we confirmed the background level of GGPP synthase activity of wild-type B. stearothermophilus FPP synthase because avian liver FPP synthase is known to have a weak GGPP synthase activity(32) . E. coli DH5alpha containing pACYC-IB was transformed with pEX11 or pFPS, which expressed wild-type FPP synthase. The color of both transformed cells remained white. These data showed that the GGPP synthase activity of wild-type FPP synthase was, if any, negligible in vivo. Then, cells carrying pACYC-IB were transformed with both plasmids derived from the libraries of 0.25 and 1 M NaNO(2) treatments. Approximately 16,700 and 7,600 recombinants from both libraries were screened. As a result, 1 and 10 red colonies were obtained, respectively. Four positive colonies were isolated from the library derived from 1 M NaNO(2) treatment, and then the pTV118N derivatives (pMU1, pMU2, pMU3, and pMU4) were isolated from the clones.

Purification of Mutated FPP Synthase

Cells of E. coli DH5alpha without pACYC-IB were transformed with pMU1, pMU2, pMU3, and pMU4 and cultured. Crude enzyme solutions were prepared from the cultures after induction with isopropyl-1-thio-beta-D-galactopyranoside. Since the B. stearothermophilus FPP synthase is thermostable, the cell homogenate was heated at 55 °C for 60 min prior to the enzyme assay to distinguish the thermostable FPP synthase from thermolabile prenyltransferases derived from the host cell. After heat treatment, the amounts of mutant FPP synthases were determined by SDS-polyacrylamide gel electrophoresis (12.5%, data not shown). The supernatant fractions contained almost all of FPP synthase although the majority of the proteins derived from host cells were precipitated. These data indicated that all mutant FPP synthases were thermostable. The specific activities of the heat-treated enzymes, which were determined by measurement of radioactivity in 1-butanol extractable materials (FPP and GGPP) of the reaction using GPP as a primer substrate, were different from each other (Table 1) and lower than that of wild-type(14) . Each of the mutated FPP synthases was purified homogeneously (Table 1). The expressed amount, total activity, and specific activity of mutant enzymes were different from each other. Especially, the total activity, which includes activities of FPP synthase and GGPP synthase, of mutant 1 and 4 were quite low. As shown in the next section, when DMAPP or GPP is used as an allylic substrate the mutated FPP synthases 1 and 4 produce GGPP with less amount of FPP. On the other hand, mutated FPP synthases 2 and 4 mainly produce FPP with less amount of GGPP. These results might indicate the toxicity of GGPP in E. coli and the existence of mechanism to suppress the over-expression of GGPP synthase.

Identification of GGPP Synthase Activity

Although red colonies of E. coli co-transformant indicated that the mutated FPP synthase produced GGPP, GGPP might be a minor component and/or products longer than GGPP might also be produced. Therefore, we first confirmed that the crude enzymes had the activity to form GGPP (data not shown). In order to characterize the mutants more precisely, the reaction products of the purified enzymes were analyzed using various allylic substrates ( Fig. 2and Table 2). In these experiments, 0.417 unit of mutated FPP synthases, 25 nmol of IPP, and 25 nmol of allylic substrate were used. One unit of enzyme activity was defined as the activity required to incorporate 1 nmol of [1-^14C]IPP into products when the assay was carried out using GPP as an allylic substrate. When FPP was used as an allylic substrate, all mutants catalyzed the condensation of IPP to produce GGPP (Fig. 2A), although the relative amounts of GGPP production were different from each other. Mutants 1 and 4 produced larger amounts of GGPP. On the other hand, the amount of GGPP produced by mutant 2 was quite small. The stereochemistry of the newly formed double bond of GGPP was also determined by normal phase thin-layer chromatography. As shown in Fig. 2B, all products were (all-E)-GGPP, indicating that the mutation did not alter the stereochemistry of reaction. When GPP was used as a allylic substrate, the patterns of product distributions from the mutant FPP synthase reactions were different (Fig. 2C). Both mutants 1 and 4 produced GGPP as a main component, and the ratios of GGPP/FPP were 4.1 and 4.6, respectively. On the other hand, the main products of mutants 2 and 3 were FPP. Especially, GGPP production of mutant 2 was quite low. The ratios of GGPP/FPP of mutants 2 and 3 were 0.28 and 0.26, respectively. In the case of using DMAPP, no significant amount of GPP was detected in any mutants (Fig. 2D), but the patterns of product distribution were almost similar to those in the case of using GPP except for the ratio of GGPP/FPP in mutant 2. In mutant 2, the ratio GGPP/FPP from DMAPP was 0.024, while that from GPP was 0.28. These data indicate that DMAPP inhibits the chain elongation from FPP to GGPP.


Figure 2: TLC autoradiochromatograms of the alcohols obtained by enzymatic hydrolysis of the products formed by the mutated FPP synthase. Panels A and B, the sample from incubation of [1-^14C]IPP and FPP with the indicated pure enzyme was analyzed by reversed phase LKC-18 TLC (A) and normal phase Kieselgel 60 TLC (B) as described under ``Experimental Procedures.'' Panel C, the sample from incubation of [1-^14C]IPP and GPP with the indicated pure enzyme was analyzed by reversed phase LKC-18 TLC. Panel D, the sample from incubation of [1-^14C]IPP and DMAPP with the indicated pure enzyme was analyzed by reversed phase LKC-18 TLC. Spots of authentic standard alcohols: GOH, geraniol; FOH, farnesol; GGOH, geranylgeraniol. Ori., origin; S.F., solvent front.





Characterization of the Mutants

Kinetic constants of the purified FPP synthase mutants for IPP, DMAPP, GPP, and FPP were determined by measuring the radioactivities in the 1-butanol-extractable products (Table 3). In these experiments, we first used 25 µM allylic substrate and 25 µM IPP as the fixed substrate for the determination of kinetic constants of IPP and allylic substrate, respectively. These concentrations were the same as those as previously reported(27) . However, when Michaelis constants were greater than 25 µM, kinetic experiments were conducted using substrates of concentration high enough for K(m) to be determined. The concentrations of countersubstrates are indicated in Table 3. The kinetic constants, V(max) and K(m), of the four mutants were different from those of wild-type, and each mutant showed unique properties.



In the case of mutant 1, the K(m) for DMAPP was larger than that of wild-type, and the V(max) was much smaller than that of wild-type, indicating that it is difficult for mutant 1 to accept DMAPP as a primer substrate. The K(m) for GPP also increased, but the decrease of V(max) was less marked than that for DMAPP. GPP was a better substrate than DMAPP. The K(m) for FPP was the smallest among the three allylic substrates and V(max)/K(m) for FPP was the largest. These data show that the activity of allylic substrates increases in the order of DMAPP, GPP, and FPP. The observation in the product analysis of mutant 1 that the amounts of GPP and FPP were smaller than that of GGPP (Fig. 2, C, lane 1 and D, lane 1) seems to reflect the activities of the allylic substrates. The kinetic constants for IPP were independently determined using DMAPP, GPP, and FPP as countersubstrates. The K(m) values for IPP were 265 µM (countersubstrate, DMAPP), 12.4 µM (GPP), and 9.40 µM (FPP). The values were dramatically different depending on the allylic substrate to be used as a co-substrate. These data suggest that the affinity of the enzyme to IPP depends upon the structure of allylic substrate. The K(m) values and V(max)/K(m) for IPP also indicate that this mutation alters the catalytic properties so that the enzyme accepts IPP and FPP as preferable substrates.

In the case of mutant 2, the K(m) for DMAPP became larger than that of wild-type, whereas the K(m) of GPP decreased. These results indicate that the affinity for allylic substrates slightly changes in such a way that the enzyme prefers GPP to DMAPP. However, the K(m) for FPP is larger than that of GPP, which is different from the case of mutant 1. The V(max) for FPP was smaller than that for GPP or that for FPP in mutant 1. Therefore, the formation of GGPP by mutant 2 seems to be less than that of FPP (Fig. 2, C, lane 2 and D, lane 2). The K(m) for IPP was also dependent on the allylic substrate employed, although the dependence was less marked than that of mutant 1. Mutant 2 shows a smaller K(m) value for GPP than for DMAPP or FPP. In addition, the K(m) value for IPP is the smallest when the co-substrate is GPP. These results indicate that the combination of IPP and GPP is the most acceptable for this mutant enzyme.

In the case of mutant 3, the K(m) for DMAPP was greater than that of wild-type, and the V(max) for DMAPP was smaller than that of wild-type. The K(m) for GPP was slightly smaller than that of wild-type and the smallest among those of the allylic substrates. In comparison with mutant 2, the K(m) values for both allylic substrates and IPP were greater than those of mutant 2 except for the K(m) value of IPP in the reaction with GPP. When GPP was used, the K(m) of IPP was the smallest among all cases.

The profile of K(m) values for both allylic substrates and IPP in mutant 4 was similar to that in mutant 1. As pointed out in the following paragraph, there is the same amino acid substitution in both mutants 1 and 4. Therefore, it seems reasonable to conclude that this substitution is essential for the formation of GGPP. However, most of the V(max) values of mutant 4 were lower than those of mutant 1, whereas most of the K(m) values of mutant 4 were lower than those of mutant 1. Although the different substitution in mutants 1 and 4 brought about the change in kinetic properties, it caused no significant change in product distribution.

Determination of the Amino Acid Sequences of the Mutants

Table 4presents sequence analyses of the mutant FPP synthases. All mutants had several mutations in the nucleic acid sequences, although the conditions in the chemical treatment for random mutation were slightly weaker than that reported previously (28) . Mutant 1 had three nucleic acid changes, in which two substitutions resulted in amino acid alterations (Y81H, L275S). Mutant 2 contained two coding mutations resulting in L34V and R59Q. Mutant 3 contained two coding mutations (V157A, H182Y) and two silent mutations. Mutant 4 possessed three coding mutations (Y81H, P239R, A265T). These mutations were distributed in the whole sequence of FPP synthase gene.



Determination of the Essential Amino Acid Residues

In order to identify the essential mutations that affect the chain length of the product, several recombinants were constructed by replacing wild-type gene fragments with the corresponding mutant fragments excised with restriction enzymes including NcoI, SnaBI, EagI, NruI, and HindIII. E. coli DH5alpha containing pACYC-IB was transformed with each of the recombinant plasmids, and then the color of the cells was determined (Table 5). From pMU1, two plasmids were made. pMU1A, which contained an N-terminal region, has a Y81H amino acid substitution. The cells transformed with this plasmid showed red color, indicating that the tyrosine at position 81 is important for determination of the chain length of the product. On the other hand, the cells transformed with pMU1C, which contained a C-terminal region, showed white color. In the case of mutant 3, the cells transformed with pMC3B, which contained two amino acid substitutions (V157A, H182Y), looked red. The transformation of pMU3A and pMU3C, both of which only contained silent mutations, did not alter the color of the cells. In the case of mutant 4, the cells transformed with pMU4A, which contained Y81H substitution as with pMU1A, formed red colonies. Transformation with pMU4C, which contained two amino acid substitutions (P239R, A265T), did not change the color of the cells.



In experiments described above, we determined the amino acid substitutions that are required for GGPP synthase activity in mutants 1 and 4. Next, we tried to determine the essential amino acid of mutations 2 and 3. The four recombinants, L34V, R59Q, V157A, and H182Y, which each contained a single amino acid substitution, were made by the Kunkel method, and then E. coli harboring pACYC-IB was transformed with each of the recombinants. Two (L34V, V157A) of the four mutants produced red colonies and the other made white colonies (Table 5). However, the red color of the colonies derived from L34V was lighter than that of pMU2. Hence, replacement of Arg-59 with Gln, which was also observed in mutant 2, might contribute to the change in the chain length of the product. These observations clearly show that single amino acid substitutions alter the ultimate chain length of the product that is determined intrinsically by individual prenyltransferase.


DISCUSSION

We tried to convert FPP synthase to GGPP synthase by using random mutagenesis and phenotypic screening. An essential element of this strategy is the efficient identification of mutants of interest among a large population of variants. For this purpose we used the red-white screening system that utilized phytoene synthase (crtB) and phytoene desaturase (crtI) to visualize the formation of GGPP in vivo. Eleven mutants that formed red colonies were selected from 24,300 clones. Four of them were analyzed in detail by sequencing the gene and determining the essential amino acid residues, reaction products, and kinetic constants. These mutants showed different properties. Characteristics of each mutant are summarized below.

Mutant 1 contained two amino acid alterations (Y81H, L275S). Y81H was found to be essential for expression of GGPP synthase activity. This enzyme mainly produced GGPP when any of the allylic substrates were used as a priming substrate, and it showed the highest GGPP synthase activity among the four mutants. Both of the K(m) values for DMAPP and GPP were increased as compared with those of wild-type. Especially, the increase of K(m) for DMAPP was prominent. The Michaelis constant for allylic substrate became smaller as the chain length of the allylic substrate increased. Consequently, FPP showed a higher affinity than DMAPP or GPP. The V(max) for DMAPP and GPP dropped. The K(m) values for IPP depended on the primer substrate employed, being decreased as the chain length of the primer substrate became longer.

Mutant 2 contained two amino acid alterations (L34V, R59Q), and L34V was essential for the activity of GGPP synthesis. This enzyme produced a small amount of GGPP with a large amount of intermediate products, reflecting the low V(max) value for FPP. The ratio of GGPP/FPP depended on the allylic substrate used as a primer. The formation of GGPP from GPP was 10 times as much as that from DMAPP. The K(m) for DMAPP was increased by this mutation, whereas the K(m) for GPP was decreased. Moreover, the K(m) value for IPP in the reaction with GPP as a primer was the smallest among those in the reaction with the three allylic substrates, and the V(max) value for IPP was the greatest when GPP was used as a primer. These data indicate that GPP is the best substrate for mutant 2.

Mutant 3 contained two amino acid alterations (V157A, H182Y), and V157A was essential for the GGPP synthase activity. The activity was weaker than those of mutants 1 and 4, and slightly stronger than that of mutant 2. When DMAPP was used, the formation of GPP was quite low, and the ratio of GGPP/FPP, which was 0.24, was similar to that obtained from the reaction using GPP as an allylic substrate.

Mutant 4 contained three amino acid alterations (Y81H, P239R, A265T), and Y81H was essential for amino acid alteration, which was also observed in mutant 1. The pattern of the product distribution was similar to that of mutant 1. However, the kinetic constants slightly differed from those of mutant 1. Mutations of P239R and/or A265T seem to affect the kinetic constants.

By analyzing kinetic properties of the mutants, we revealed several factors that might be related to the change from FPP synthase to GGPP synthase. They include decrease of total reaction rate, increase of K(m) (DMAPP), and change of K(m) (IPP) depending on an allylic substrate used as a primer. Detailed mechanisms and kinetic constants of prenyltransferases have been discussed only for the single condensation of GPP and IPP catalyzed by avian FPP synthase(33, 34) . The FPP synthase has been shown to obey the ordered sequential mechanism for synthesis of FPP from IPP and GPP as shown in . The steady state kinetic constants can be expressed in terms of individual rate constant for

where

Release of FPP is rate-limiting for condensation of IPP and GPP, where k(5) approx 50k(6), and V(max) approx k(6)[Et]. The slight decrease in V(max) seen for the mutants using GPP and IPP as substrates could result from a few hundredfold decrease in the rate of chemical step (k(5)), with a concomitant change in the rate-limiting step, or from an additional slight reduction in product release step (k(6)). If the chemical step becomes rate-limiting, the decrease of k(5) will bring about an accumulation of GPP in the reaction between DMAPP and IPP. However, the mutants did not accumulate GPP (Fig. 2). Moreover, it is unlikely that the decrease of the chemical step rate results in the formation of longer chain products. Therefore, the decrease of V(max) seems to result from that of k(6). If this hypothesis is true, the decrease of V(max) means the increase of the affinity between FPP and enzyme. Actually, in mutants 1 and 4, when the reaction was started with IPP and GPP, the amount of FPP as the intermediate was low. All mutants showed affinities for FPP to yield GGPP. It is obvious that the affinity between FPP and the enzyme increases. Moreover, these results might indicate that the mutation of a prenyltransferase so as to produce a prenyl product with a longer chain length than that of the original enzyme is accompanied by sacrifice of the total activity, which is related to k(6).

It has been well known that the prenyltransferases that produce long chain prenyl diphosphates such as hexaprenyl diphosphate, solanesyl diphosphate, and undecaprenyl diphosphate do not accept DMAPP as a priming substrate but need to use GPP, FPP, or GGPP(35, 36, 37, 38) . The mutants show decreased activities for DMAPP. This tendency is prominent in mutants 1 and 4 and is similar to the case of the long chain prenyltransferases described above.

In all mutants, the K(m) values for IPP are dependent on the allylic substrate that is used as a primer substrate. Especially, in mutant 1, the K(m) values for IPP using DMAPP, GPP, and FPP are 265 ± 3.50, 12.4 ± 0.80, and 9.40 ± 1.10 µM, respectively. From the , the increase of K(m) is brought about not only by the increase of K(d) but also by the decrease of k(5). In mutants 1 and 4, there is no significant difference between the V(max) values for DMAPP and GPP. Therefore, it does not seem to be suitable to assume that k(5) contributes to the change of K(m). If the difference of K(m) is mainly due to the difference of K(d), this phenomenon indicates that a single enzyme shows different affinities to the same substrate depending on countersubstrates. These results might indicate that the binding of allylic substrate to prenyltransferase causes a conformational change that affects the affinity of IPP. Moreover, during the consecutive reaction of prenyltransferase, a series of conformational changes might occur, and the changes might be essential for the prenyltransferase reaction. At present, it is unclear which of the two scenarios is suitable.

We found that the single alteration, Y81H, L34V, or V157A, caused a change of the ultimate product. How are these amino acids involved in catalytic activity? The structural genes for a considerable number of prenyltransferases have been identified and characterized. Comparisons of amino acid sequences of the prenyltransferases were reported by Koyama et al. (14) and Chen et al.(18) , who indicated 7 conserved regions (a-g) and 5 conserved regions, respectively. The conserved regions (a-g), which are proposed by Koyama et al.(14) are indicated in the sequence alignment of chick and B. stearothermophilus FPP synthase (Fig. 3). Leucine at position 34 of B. stearothermophilus FPP synthase is located upstream of region a, which contains the highly conserved GKXXR motif. Tyrosine at position 81 situates in region b, which contains the first DDXXD motif. Prenyltransferases have two conserved DDXX(XX)D aspartate-rich motifs, which are assumed as binding sites for the diphosphate moieties of IPP and the allylic substrates. Valine at position 157 is located in front of region d, which contains the conserved GQXXD motif. However, no significant similarity or difference at the three mutated positions on sequence alignment of prenyltransferases has been observed so far.


Figure 3: The comparison of the secondary structure of avian FPP synthase with the predicted secondary structure of B. stearothermophilus FPP synthase. The comparisons of both the primary and secondary structures of avian and B. stearothermophilus FPP synthases are shown. In the primary structure, the regions (a, b, c, d, e, f, and g) that have been reported by Koyama et al. (14) to show significant sequence conservation are boxed. The essential amino acids, which are related to chain length determination, are indicated by an underline, and the substituted amino acids are indicated below. The secondary structure of avian FPP synthase is cited from Tarshis et al.(41) and indicates above the primary structure. The letters (A, B, C, D, E, F, G, H, I, J, alphaI, alphaII, alphaIII) indicated above the secondary structure of avian FPP synthase are the helix names reported by Tarshis et al.(41) . The secondary structure drawn below the primary structure of B. stearothermophilus FPP synthase is essentially predicted by the methods of Chou and Fasman (39) and Robson (40) .



The secondary structure of B. stearothermophilus FPP synthase around Tyr-81 was predicted as alpha-helix by the methods of Chou and Fasman (39) and of Robson (40) (Fig. 3). This secondary structure was also supported by the crystal structure of avian FPP synthase (Fig. 3)(41) . If the region forms alpha-helix, the distance between Tyr-81 and Asp-86, which is the first aspartate of the first DDXXD motif, is 11 12 Å. The first DDXXD motif has been thought to bind a diphosphate moiety of an allylic substrate via magnesium ion. The distance is almost the same as the length of the hydrocarbon moiety of FPP. These results might suggest that the side chain of the amino acid blocks further condensation beyond FPP by direct contact with the hydrocarbon moiety of FPP. Further analysis of these amino acid residues (Tyr-81, Leu-34, and Val-157) is necessary to reveal the precise role of the substituted amino acids and, finally, the mechanism of the chain length determination.

Recently, Chen et al.(18) have proposed a phylogenetic tree for isoprenyl diphosphate synthases based on a comparative primary structures. In the tree, the most distant separation is between hexaprenyl diphosphate synthase and other synthases. Farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families. The separation between FPP synthase and GGPP synthases occurs after the separation between kingdoms. They have postulated that GGPP synthase that produced FPP as an intermediate product is an ancient enzyme based on the phylogenetic tree and the property of archaebacterium Methanobacterium thermoautotrophicum GGPP synthase, which is bifunctional enzyme to produce FPP and GGPP(42) . Our conversion from FPP synthase to GGPP synthase, which also shows the activity of FPP synthase, can be a retrospection of prenyltransferase evolution.


FOOTNOTES

*
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
To whom correspondence should be addressed. Tel.: 22-217-7272; Fax: 22-217-7293; sohnuma{at}seika.che.tohoku.ac.jp (for S.-I. O.). Tel.: 22-217-7270 (for T. N.).

(^1)
The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate.


ACKNOWLEDGEMENTS

We are grateful to Kazutake Hirooka for cooperation in the characterization of the GGPP synthase. We thank Dr. Norihiko Misawa, Kirin Brewery Co., Ltd., for providing the E. uredovora crt genes. We thank Dr. Chikara Ohto and Ayumi Koike-Takeshita, Toyota Motor Corp., for the helpful discussions.


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