Molecular Cloning, Expression, and Purification of Undecaprenyl Diphosphate Synthase
NO SEQUENCE SIMILARITY BETWEEN E- AND Z-PRENYL DIPHOSPHATE SYNTHASES*

Naoto Shimizu, Tanetoshi KoyamaDagger , and Kyozo Ogura

From the Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai 980-8577, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of the gene for undecaprenyl diphosphate synthase was successful, providing the first primary structure for any prenyltransferase that catalyzes Z-prenyl chain elongation. A genomic DNA library of Micrococcus luteus B-P 26 was constructed in Escherichia coli, and the recombinant clones were grown on nylon membranes. The membrane was incubated directly by floating it on a reaction mixture containing radiolabeled isopentenyl diphosphate, nonlabeled farnesyl diphosphate, and Mg2+. Only the clones harboring plasmids encoding prenyltransferases could take up the substrates to synthesize and accumulate radiolabeled products inside the cells in amounts large enough to be detectable by autoradiography. Four positive colonies were found among about 4,000 bacterial colonies of the genomic DNA library. Two of them carried the gene for undecaprenyl diphosphate synthase, which catalyzes the Z-prenyl chain elongation, and the others carried the (all-E)-hexaprenyl diphosphate synthase genes (hexs-a and hexs-b; Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Bacteriol. 180, 1578-1581). The undecaprenyl diphosphate synthase, which had a predicted molecular mass of 28.9 kDa, was overproduced in E. coli cells by applying a soluble expression system, and it was purified to near homogeneity. The deduced primary structure of the Z-prenyl chain-elongating enzyme is totally different from those of E-prenyl chain-elongating enzymes, which have characteristic conserved regions, including aspartate-rich motifs.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

In the biosynthesis of isoprenoid compounds, including sterols, respiratory quinones, carotenoids, glycosyl carrier lipids, natural rubber, and prenyl proteins, all of these compounds are derived from linear prenyl diphosphates, which are synthesized by sequential condensations of isopentenyl diphosphate (IPP)1 with allylic prenyl diphosphates. These condensations are catalyzed by a family of prenyltransferases.

During the last decade the structural genes for many kinds of prenyltransferases that catalyze E-type prenyl chain elongation have been cloned and characterized (1). Multiple alignments of the deduced amino acid sequences of the E-type prenyl chain-elongating enzymes, including farnesyl diphosphate synthase (FPS), geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase (HexPS), heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase, and decaprenyl diphosphate synthase, have shown the presence of two characteristic aspartate-rich DDXXD motifs and several other conserved regions in the primary structures of prenyltransferases (2-4). X-ray crystallography (5) and site-directed mutagenesis studies of FPS (6-15) and geranylgeranyl diphosphate synthase (16, 17) have revealed the precise mechanisms of prenyl chain elongation and determination of the product chain length.

On the other hand, nothing is known about the structure of Z-prenyl chain-elongating enzymes. Consequently, only limited information is available about the molecular mechanism of enzymatic Z-prenyl chain elongation.

In bacteria, undecaprenyl diphosphate synthase (UPS) catalyzes the Z-prenyl chain elongation onto (all-E)-farnesyl diphosphate (FPP) as a primer to yield undecaprenyl diphosphate (UPP), a C55-prenyl diphosphate with E,Z-mixed stereochemistry (18, 19), which is the direct precursor of the glycosyl carrier lipid in the biosynthesis of cell wall polysaccharide components such as peptidoglycan and lipopolysaccharide.

This paper describes the first gene cloning and overproduction of UPS

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [1-14C]IPP (1.95 TBq/mol) was purchased from Amersham Pharmacia Biotech. Nonlabeled IPP, DMAPP, FPP, and Z-GGPP were synthesized according to the procedure of Davisson et al. (20). Restriction enzymes, T4 DNA ligase, and Ex Taq DNA polymerase were purchased from Takara Shuzo Co., Ltd. Ribonuclease and acid phosphatase were purchased from Sigma Chemical Company. All other chemicals were of analytical grade.

Bacterial Strains, Plasmids, and Media-- Micrococcus luteus B-P 26 was used as the source for chromosomal DNA. Escherichia coli JM109 was used as the host for the M. luteus B-P 26 DNA library. The plasmids pFP00 and pHX06, which carry the genes for FPS and HexPS of M. luteus B-P 26, respectively, were described in our recent paper (21). LB broth (10 g of Bacto-tryptone, 5 g of yeast extract, and 10 g of NaCl dissolved in 1 liter of water), supplemented with 50 µg/ml ampicillin, where appropriate, was used to propagate bacterial strains unless otherwise stated. The pET expression system was obtained from Novagen, Inc.

General Methods-- Restriction enzyme digestions, transformations, and other standard molecular biology techniques were carried out as described by Sambrook et al. (22).

Identification of Colonies Overexpressing Extra Prenyltransferases in E. coli Cells by Colony Autoradiography-- The procedure was essentially similar to that developed by Raetz (23). On a sterilized nylon membrane (0.2-µm grid of the mesh Biodyne A, 82-mm diameter, Pall), which had been placed on an LB-agar plate containing ampicillin, 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside, and isopropyl-beta -D-thiogalactopyranoside (IPTG), E. coli transformants were inoculated and incubated at 37 °C for 12 h. Several sheets of the replica filter were prepared by filter-to-filter contact followed by incubation at 37 °C for another 6 h on a fresh LB-agar plate containing ampicillin to propagate bacterial cells. The master filter was also laid on a fresh LB-agar plate containing ampicillin, incubated at 37 °C for another 3 h, and then kept at 4 °C until use. Each of the replica filters was removed from the plate, floated on a 0.6-ml TE buffer solution (10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA) in a plastic Petri dish lid, keeping the colony side up to wash out residual nutrient broth absorbed in the filter. Then the filters were placed on a paper towel to remove the water. The incubation mixtures for the direct assay of the replica filter contained, in a final volume of 0.6 ml, 100 mM Tris-HCl buffer, pH 7.5, 5 mM MgCl2, 5 µM DMAPP, 0.46 µM [1-14C]IPP (specific activity, 1.95 TBq/mol) (Conditions I) or 100 mM Tris-HCl buffer, pH 7.5, 0.5 mM MgCl2, 50 mM KCl, 5 µM FPP or Z-GGPP, and 0.46 µM [1-14C]IPP (Conditions II). The replica filter was floated on one of the assay mixtures in a Petri dish keeping the colony side up. Then the Petri dishes were placed in a sealed plastic bag with a wetted paper towel and incubated at 37 °C for 6 h. After incubation, the filters were placed on a paper towel and kept at room temperature for 30 min to remove water from the filter. The initial concentration of [1-14C]IPP was set up low enough to reduce background signals from unreacted substrates without washing them out from the filter. The filters were then wrapped with Saran Wrap and exposed on a Fuji film BAS-IIIs imaging plate at room temperature. The distributions of radioactivity in the filters were analyzed with a Fuji BAS 1000 Mac bioimaging analyzer to detect positive colonies.

Genomic DNA Preparation-- M. luteus B-P 26 grown in 2 liters of L (Lennox) broth (10 g of Bacto-tryptone, 5 g of yeast extract, 5 g of NaCl, and 1 g of glucose dissolved in 1 liter of water) at 30 °C was harvested in the late log phase, and the chromosomal DNA was prepared as described by Saito and Miura (24).

Construction of M. luteus B-P 26 Genomic DNA Library-- Partial digestion of the chromosomal DNA (72 µg) was carried out with Sau3AI (128 units). DNA fragments 4-8 kilobases long were extracted from 0.8% agarose gel after electrophoresis. The DNA fragments were ligated into the BamHI site of pUC119 with T4 DNA ligase, and the ligation mixture was used directly for transformation of E. coli JM109 to construct a genomic DNA library of M. luteus B-P 26.

Screening of the M. luteus B-P 26 Genomic DNA Library-- An appropriate amount of E. coli cells carrying the genomic DNA library was inoculated (4,000 colonies/plate) on a nylon membrane (0.2-µm grid of the mesh Biodyne A, 132-mm diameter) that had been laid on an LB-agar plate containing ampicillin. After incubation at 37 °C for 14 h, two replica filters were made by filter-to-filter contact. The replica filters were incubated for another 7 h on new LB-agar plates containing ampicillin. Then, UPS activity was screened over the library by the colony autoradiography method as described above using FPP or Z-GGPP as a primer substrate. In this case, the total volume of the reaction mixtures was increased to 1.6 ml adjusting to the size of the filter. After seeking the coincident spots in the autoradiograms of the duplicate filter, double-positive clones were isolated from the master plate and analyzed further.

Preparation of Cell-free Homogenates of E. coli Transformants-- Transformed E. coli cells were cultured in 2 ml of LB broth at 37 °C overnight. The cells were harvested, resuspended in 0.3 ml of TE buffer, and homogenized by sonication. The homogenate was centrifuged at 10,000 × g for 2 min, and the supernatant was examined for prenyltransferase activity.

UPS/HexPS Assay-- The incubation mixture contained, in a final volume of 0.2 ml, 100 mM Tris-HCl buffer, pH 7.5, 0.5 mM MgCl2, 50 mM KCl, 5 µM FPP or Z-GGPP, 0.46 µM [1-14C]IPP, 0.05% (w/v) Triton X-100 (for UPS assay), and crude cell-free homogenate (1 µg of protein) of an E. coli transformant to be examined. The mixture was incubated at 37 °C for 1 h and then treated with 1-butanol to extract the product of the prenyltransferase reaction. The radioactivity in the butanol extract was measured with an Aloka LSC-1000 liquid scintillation counter.

Product Analysis of Prenyltransferases-- After incubation, the radioactive prenyl diphosphates in the reaction mixture were hydrolyzed to the corresponding alcohols with potato acid phosphatase according to our method reported previously (25). The alcohols were extracted with pentane and analyzed by thin layer chromatography (TLC) on a reversed phase LKC-18 plate (Whatman) with a solvent system of acetone/water (19:1). Normal phase TLC of the radioactive prenyl diphosphate products was also carried out on a Silica Gel G-60 plate (Merck) with a solvent system of 1-propanol, 29% aqueous NH3, H2O (6:3:1). The positions of authentic standards were visualized with iodine vapor, and the distribution of radioactivity was determined by autoradiography with a bioimaging analyzer.

DNA Sequence Analysis-- All DNA sequences were determined by the dideoxy chain termination method with a DNA sequencer (LI-COR model 4000L). Computer analysis and comparison of DNA sequences were performed using GENETYX genetic information processing software (Software Development).

Overproduction of UPS Gene Product in E. coli-- The UPS gene sequence was amplified by polymerase chain reaction from M. luteus B-P 26 genomic DNA, using the primers 5'-GAGGTTGACATGTTTCCAATTAA-3' and 5'-CATGATTTAAAGCTTATAATCCACC-3'. These primers were designed so that the amplified DNA would contain the restriction endonuclease sites for AflIII and HindIII at its 5'- and 3'-ends, respectively. The resulting polymerase chain reaction products were digested with AflIII and HindIII, purified, and ligated into the pET22b expression vector. The resultant vector, designated as pET22bMLU, was used to transform E. coli BL21(DE3) cells. The UPS gene was fused to the pelB signal sequence so that the translated protein could be secreted to the periplasmic region followed by removal of the signal peptide by endogenous signal peptidase. Recombinant clone was selected for the UPS gene insert, and a single colony was grown in 3 ml of LB medium for 9 h. The resulting culture was inoculated into 2 liters of M9-glucose (1 mg of thiamine, 120 mg of MgSO4, 11 mg of CaCl2, and 2 g of glucose in 1 × M9 salts (22)) plus ampicillin (50 mg/liter) and grown at 37 °C. The cell growth was monitored and induced with 1.0 mM IPTG when cells reached 0.3 absorbance unit at 600 nm. The cells were grown for another 26 h and harvested by centrifugation.

Purification of UPS-- The cell pellets (3 g, wet weight) of BL21(DE3)/pET22bMLU were suspended in 30 ml of TE buffer and disrupted by sonication (Fraction I). The extracts from the cells were collected by ultracentrifugation, and the supernatant (Fraction II) was subjected to further purification. The protein fraction precipitated from the 100,000 × g supernatant by 30-60% saturation with ammonium sulfate was dissolved in 30 ml of 20 mM Tris-HCl buffer, pH 7.3 (buffer A) (Fraction III). Then the UPS solution was applied to a Mono Q HR 10/10 column (Amersham Pharmacia Biotech) equilibrated with buffer A. Proteins were eluted at 1 ml/min with a stepwise gradient of 0-0.5 M NaCl in buffer A. The UPS fractions (Fraction IV) after Mono Q chromatography were subjected to gel filtration performed with Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) at 0.4 ml/min with 50 mM phosphate buffer, pH 7.0, containing 150 mM NaCl (buffer P). All purification steps were carried out at 4 °C. Protein elution was monitored by measuring absorbance at 280 nm.

Amino-terminal Sequence Analysis of UPS-- The 30-kDa protein (30 µg) purified by Mono Q chromatography was developed on a 15% SDS-polyacrylamide gel, and then electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad). The transferred polypeptide was visualized by Coomassie Brilliant Blue and sequenced using the HP G1005A protein sequencing system.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Determination of Screening Conditions-- To examine the possibility of selective labeling of E. coli cells that produce recombinant prenyltransferase, we incubated intact colonies on a nylon membrane with radiolabeled substrates.

Three recombinant clones, JM109/pFP00 producing FPS of M. luteus B-P 26 (24), JM109/pHX06 producing HexPS of M. luteus B-P 26 (21), and JM109/pUC119 as a control, were streaked separately on a nylon membrane placed on an agar plate. Six replica filters were prepared and were subjected to prenyltransferase activity assays by the colony autoradiography method with three different sets of substrates.

If nonspecific adsorption or absorption of radioactive IPP by E. coli cells or by the membrane filter had occurred primarily, all of the colonies should have given nonselective signals, or the background level should have been high in the autoradiogram. In fact, however, the clone expressing a certain level of prenyltransferase was labeled selectively enough to give distinct signals in the autoradiograms after incubation with radiolabeled substrates (Fig. 1). It was thus found to be possible to discriminate the three kinds of clones by colony autoradiography.


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Fig. 1.   Autoradiograms of the replica plates of the colonies of E. coli transformants harboring pFP00, pHX06, or pUC119. Six replica filters of the E. coli colony streaks harboring expression plasmids for prenyltransferases were prepared and incubated by being floated on the two series of assay mixtures of Conditions I (panels a--c). Conditions I are shown with 0.5 mM MgCl2 (b), except that DMAPP was omitted (a), and except that DMAPP was replaced with FPP (c). Panels d--f, Conditions I with 5 mM MgCl2 (e), except that DMAPP was omitted (d), and except that DMAPP was replaced with FPP (f) as described under "Experimental Procedures." The positions of the transformants are indicated in each plate.

When the Mg2+ concentration in the incubation mixture was 0.5 mM (Conditions II, Fig. 1, a-c), specific selection of JM109/pHX06, which expresses HexPS, was successful when assayed with the mixture containing FPP as the priming substrate (Fig. 1c). No colony was labeled when [1-14C]IPP was employed as the sole substrate (Fig. 1a). The colonies expressing FPS, JM109/pFP00, could not be detected clearly with its substrate DMAPP as the allylic primer (Fig. 1b). However, strong signals were obtained from the colonies of JM109/pFP00 with DMAPP (Fig. 1e), when the Mg2+ concentration as raised to 5 mM (Conditions I; Fig. 1, d-f). This is consistent with the observation that FPS requires a higher Mg2+ concentration than HexPS (26). In this case, discrimination between JM109/pUC119 and JM109/pFP00 was possible, but JM109/pHX06, which should not utilize DMAPP as a primer, also gave strong signals (Fig. 1e). This was probably because of a low but significant activity of the host FPS, which could produce FPP acceptable as substrate for HexPS under these conditions. The case was similar with JM109/pFP00 (Fig. 1d). The JM109/pFP00 colonies were visualized under the conditions where [1-14C]IPP was the only substrate, probably because of the production of DMAPP by the action of IPP isomerase of the host cells. Even with such an undesirable background of prenyltransferase activity by the host cells, this assay method appears promising for screening a clone expressing a prenyltransferase if the specific assay conditions are chosen properly .

Isolation of the Gene for UPS-- We first applied this method to screening a transformant expressing UPS of M. luteus B-P 26. Based on the fact that the UPS activity can be detected by using FPP as the allylic primer substrate and that the optimum concentration of Mg2+ for UPS is lower than for FPS (27), it is possible to set proper screening conditions for UPS. Furthermore, because UPS can utilize Z-GGPP as a primer, which cannot be a substrate for HexPS (26, 28), it may be possible to identify the clone expressing UPS among the positive clones under the assay conditions using FPP as the priming substrate. With this expectation, about 4,000 transformants of E. coli cells carrying a genomic DNA library of M. luteus B-P 26 were screened by this filter assay method, using Conditions II with FPP or Z-GGPP as the priming substrate (Fig. 2). As a result, we could obtain four positive colonies coinciding in the two assay systems using different priming substrates. Then the four clones were isolated from the master plate, and the prenyltransferase activities in the cell-free homogenates of the clones were examined precisely as described under "Experimental Procedures."


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Fig. 2.   Autoradiograms of the replica plates of the genomic DNA library of M. luteus B-P 26. The replica filters of the genomic DNA library of M. luteus B-P 26 were incubated by floating them on the assay mixtures of Conditions II using the following substrates: A, [1-14C]IPP + FPP; and B, [1-14C]IPP + Z-GGPP as described under "Experimental Procedures." Arrows indicate the signals of double-positive clones (see "Results").

As shown in Table I, all of the four clones expressed significant levels of prenyltransferase activities when assayed with FPP as the primer, and two of them showed significant activities when Z-GGPP was the allylic substrate. TLC analyses of the butanol-extractable polyprenyl products and the corresponding alcohols obtained by phosphatase treatment clearly indicated that the two clones produced UPP and that the rest of them produced HexPP. Hence, we assigned the former two clones as transformants with plasmids carrying the UPS gene of M. luteus B-P 26 and the latter two as those carrying the genes for the two essential components of HexPS of the same bacterium, which we have cloned recently (21). The two clones expressing the UPS activity were found to carry plasmids of different insert size with overlapping fragment, and the smaller one, containing a 6-kilobase insert, was designated as pSU29S2 and was subjected to deletion experiments and sequence determination.

                              
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Table I
Prenyltransferase activities of the clones selected by the direct filter assay
Each of the cell-free homogenates of the four double-positive clones, JM109/pSU26, -/pSU27, -/pSU28, and -/pSU29S2, obtained by means of colony autoradiography, was examined for the prenyltransferase activity as described under "Experimental Procedures." Radioactivities obtained under three different sets of assay conditions using DMAPP, FPP, or Z-GGPP as the priming substrate are shown. JM109/pUC119 was assayed as a control.

Characterization of the UPS Gene-- Deletion experiments of pSU29S2 indicated that the DNA sequence responsible for the UPS activity was located within a 1-kilobase DNA region. Analysis of the nucleotide sequence of the region revealed the presence of one open reading frame of 750 base pairs. The deduced amino acid sequence of the UPS is shown in Fig. 3 with its nucleotide sequence. The primary structure of the UPS of M. luteus B-P 26 is totally different from those of the prenyltransferases (prenyl diphosphate synthases) for E-prenyl chain elongation (1).


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Fig. 3.   Nucleotide and deduced amino acid sequences of the UPS of M. luteus B-P 26. Numbers on the left refer to nucleotides; those on the right refer to amino acids. The DDXXD region is underlined. Upstream from the initiation codon, the putative promoter (-10, -35) and ribosome binding site (RBS) are also underlined.

Purification of UPS-- SDS-polyacrylamide gel electrophoresis analysis of the induction products in the whole cell lysate of the E. coli cells harboring pET22bMLU, pET22b containing the UPS gene, showed the existence of two protein forms with slightly different molecular masses of 30 and 31 kDa (Fig. 4, lane 2). After ultracentrifugation, the 30-kDa protein product was found in the soluble fraction (Fig. 4, lane 4), and the 31-kDa protein remained in the pellet fraction (Fig. 4, lane 3). The pellet fraction showed the UPS activity as well as the soluble fraction (data not shown). The UPS in the soluble fraction was readily purified to near homogeneity through ammonium sulfate precipitation and two chromatographic steps, which are summarized in Table II. Hydrophobic chromatography was not suitable for the purification of this enzyme because it was easily absorbed by butyl-, octyl-, and phenyl-Sepharose columns (Amersham Pharmacia Biotech) and eluted very broadly during decreasing linear gradient of ammonium sulfate. The ammonium sulfate precipitate fraction was applied to a Mono Q column, from which the UPS was eluted by 100 mM NaCl. As shown in Fig. 4, the UPS fraction after Mono Q chromatography gave a protein band at 30 kDa (lane 6). However, after storage at 4 °C for a week, the protein migrated at a molecular size of 29 kDa (lane 7). After gel filtration with Superdex 200, the purity of the enzyme appeared to be better than 95% (Fig. 4, lane 8).


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Fig. 4.   Purification of the overexpressed UPS. Protein samples were separated by 15% SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue R-250. Lane 1, BL21(DE3)/pET22b cell lysate after IPTG treatment (control). Lanes 2-8 show 10 µg of proteins of BL21(DE3)/pET22bMLU cells after IPTG induction: lane 2, cell lysate; lane 3, 100,000 × g pellet; lane 4, 100,000 × g supernatant; lane 5, 30-60% ammonium sulfate precipitate; lane 6, UPS fraction after Mono Q chromatography; lane 7, fractions containing UPS after storage at 4 °C for a week; lane 8, UPS fraction after Superdex 200 chromatography. M, molecular mass standards, 97.4, 66, 45, 31, 21.5, and 14.5 kDa from the top.

                              
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Table II
Purification of recombinant M. luteus B-P 26 UPS
Details of the purification procedures are described under "Experimental Procedures." Enzymatic activity was assayed at 37 °C with mixtures of 0.46 µM [1-14C]IPP, 5 µM Z-GGPP, 0.5 mM MgCl2, 50 mM KCl, and 0.05% (w/v) Triton X-100 in 100 mM Tris-HCl buffer, pH 7.5. 

Amino-terminal sequencing was performed to verify that the 30-kDa protein after Mono Q chromatography was that encoded by the pET22bMLU vector. As a result, the sequence of 20 amino acids from the amino terminus was AQPAMAMFPIKKRKAIKNNN, indicating that 6 residues from the amino terminus (AQPAMA) was the remainder of the pelB signal sequence which resulted from the cleavage at an unexpected position. The sequence MFPIKKRKAIKNNN was in complete agreement with that predicted from the DNA sequence of the UPS.

Fig. 5 shows radiochromatographic analyses of reaction products of the purified UPS from BL21(DE3)/pET22bMLU when FPP was used as the allylic primer substrate. The major prenol obtained after phosphatase treatment had the same mobility as authentic undecaprenol (C55OH) on reversed phase TLC (panel A, lane 4). Small amounts of intermediate products with chain lengths of C20-C50 were also observed. When Z-GGPP was used as the allylic primer, the TLC patterns were almost similar to those of lane 4, except that the intermediate products with chain length of C20 were not observed (data not shown). On normal phase TLC (panel B, lane 4), the major product had a larger RF value (0.48) than those of HexPP (0.42, lane 2) and FPP (0.37, lane 1), showing that it was UPP.


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Fig. 5.   TLC autoradiograms of the reaction products of the purified UPS. The products were analyzed as described under "Experimental Procedures." Panel A, reversed phase TLC of the alcohols after phosphatase treatment. Lane 1, JM109/pFP00 (produces FPP); lane 2, JM109/pHX06 (produces HexPP); lane 3, BL21(DE3)/pET22b (as control); lane 4, UPS purified from BL21(DE3)/pET22bMLU. Arrowheads indicate the positions of authentic specimens: C15, (all-E)-farnesol; C20, (all-E)-geranylgeraniol; C30, (all-E)-hexaprenol; C55, (2Z, 6Z, 10Z, 14Z, 18Z, 22Z, 26Z, 30Z, 34E, 38E)-undecaprenol; C60, (2Z, 6Z, 10Z, 14Z, 18Z, 22Z, 26Z, 30Z, 34Z, 38E, 42E)-dodecaprenol. Panel B, normal phase TLC of the reaction products. Lane 1, JM109/pFP00 (FPP); lane 2, JM109/pHX06 (HexPP); lane 3, BL21(DE3)/pET22b (as control); lane 4, UPS purified from BL21(DE3)/pET22bMLU. The small bands appearing above the major products are the corresponding monophosphates produced artificially during chromatography. Ori., origin; S.F., solvent front.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Although the reaction catalyzed by UPS is similar to those of E-type prenyl chain-elongating enzymes, we have never found a candidate gene for the Z-type prenyltransferase by searching the whole genome sequence data base of E. coli on the assumption that Z-prenyltransferases also have some homologies to the E-prenyltransferases whose genes are already cloned (29, 30). We therefore planned shotgun screening of the M. luteus B-P 26 genomic DNA library for expression of prenyltransferase activities. To screen as many clones as possible simultaneously, we conducted the colony autoradiography method originally developed by Raetz for the selection of mutants defective in specific enzymes (23). Replica filters of M. luteus B-P 26 genomic DNA library were incubated with the substrates for prenyltransferases, and enzymatic activities were screened directly on the intact colonies. The lipid bilayers of E. coli cells were thought to be impermeable to charged molecules such as IPP and FPP. However, Takatsuji et al. (31) and Fujisaki et al. (32) reported that 14C-labeled IPP was incorporated into isoprenoid compounds in the presence of Mg2+ ions when incubated with Arthrobacter or lyophilized E. coli cells. This observation suggests that E. coli cells are permeable to IPP in the presence of Mg2+. Therefore, we expected that both 14C-labeled IPP and allylic diphosphates would permeate the cells and that the E. coli transformants overexpressing prenyltransferases could utilize them to yield radioactive products in the cells in amounts large enough to detect by autoradiography. Thus, we were able to set up proper conditions for screening the UPS activity and succeeded in cloning the UPS gene.

Our first attempt to overproduce the recombinant UPS in an active form in E. coli cells failed. We ascribed the failure to the overproduction and accumulation of UPP, which might disrupt the metabolic pathway because UPP is involved in cell wall biosynthesis of the host bacterium. Then, we introduced the bacterial pelB signal sequence to the amino terminus of the UPS to localize the protein product in the periplasmic space so that interference in the host growth could be avoided. The expression of the protein was controlled by a T7 RNA polymerase-mediated system in a recombinant strain of E. coli BL21(DE3). Two major protein bands were detected on the SDS-polyacrylamide gel electrophoresis of the whole cell lysate of transformants after treatment with IPTG. Because the 31-kDa protein was detectable only in the insoluble pellet fraction, this may be attributable to the products with unprocessed pelB leader sequence encoded by the pET22b vector, which tends to associate with the membrane fraction. Sequence analysis of the 30-kDa protein product obtained in the Mono Q chromatography indicated that part of the signal sequence was left at the amino terminus of the recombinant enzyme. A signal peptidase remaining in the UPS fraction after Mono Q chromatography might have processed the residual peptide properly while kept at 4 °C for a week. SDS-polyacrylamide gel electrophoresis analysis of the purified UPS shows a single band at 29 kDa (Fig. 4, lane 8), which is consistent with the molecular weight of 28,876 calculated from the deduced amino acid sequence. Because all of the three protein species, 31-, 30-, and 29 kDa proteins, showed considerable UPS activity, it is likely that flanking amino-terminal sequence does not affect the catalytic activity.

The purified protein itself had the UPS activity, showing almost the same specific activity as reported by Muth and Allen (19) for an extensively purified specimen of UPS of Lactobacillus plantarum. This indicates that the 29-kDa protein overproduced is actually UPS and not an accessory protein that stimulates the endogenous UPS. Thus it was confirmed that the gene for the Z-prenyltransferase was first cloned and overproduced.

The E-prenyltransferases so far known have several characteristic regions well conserved regardless of the chain length of the product. The regions include two DDXXD motifs that are important for both catalytic function and substrate binding (7, 9, 12, 15). It is also shown that prenyl diphosphate is bound to one of the conserved DDXXD motifs through a magnesium bridge (15). The Z- and E-prenyltransferases are similar in that they both catalyze sequential condensations between IPP and allylic diphosphates with concomitant release of inorganic pyrophosphate in the presence of magnesium ions. In view of this similarity, it was expected that a considerable extent of sequence similarity would be found between the Z- and E-prenyltransferases. Surprisingly, however, no similarity was found between them. There is a DDXXD-like motif structure in residues 117-121 in the UPS, but the significance of this structure remains to be explored.

It is assumed that the E-farnesyl-Z-octaprenyl (undecaprenyl) moiety and the E-prenyl side chain of menaquinone are early ancestors of a variety of isoprenoid compounds because undecaprenyl phosphate and menaquinone play essential roles in cell wall construction and membrane-associated bioenergetics, respectively, in prokaryotes. Thus, the Z- and E-prenyltransferases responsible for these two isoprenoid moieties might have evolved independently. Our search through protein data bases for amino acid sequences similar to that of UPS revealed several as yet unidentified proteins, including eukaryotic proteins. The identification of these proteins will be of interest from an evolutional aspect.

It is also interesting to learn the similarity and difference in the three-dimensional structures of these two types of prenyltransferases. The stereochemical courses of Z- and E-prenyltransferase reactions are similar to each other in that in both cases the C-C bond is formed on the same side of IPP as the C-H bond that is cleaved (33). This suggests that the conformation of the IPP molecule bound in the active site of the Z-prenyltransferase must be different from that in the case of the E-prenyltransferases. A better understanding of the molecular mechanism of the UPS reaction will provide a practical strategy for designing of new antimicrobial molecules.

    FOOTNOTES

* This work was supported by Grant-in-aid for Scientific Research 09480138 from the Ministry of Education, Science, and Culture of Japan and JSPS-RFTF97I00302 from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB004319.

Dagger To whom correspondence should be addressed. Tel.: 81-22-217-5621; Fax: 81-22-217-5620; E-mail: koyama{at}icrs.tohoku.ac.jp.

1 The abbreviations used are: IPP, isopentenyl diphosphate; FPS, farnesyl diphosphate synthase; HexPS, hexaprenyl diphosphate synthase; UPS, undecaprenyl diphosphate synthase; FPP, (all-E)-farnesyl diphosphate; UPP, undecaprenyl diphosphate; DMAPP, dimethylallyl diphosphate; Z-GGPP, (Z,E,E)-geranylgeranyl diphosphate; IPTG, isopropyl-beta -D-thiogalactopyranoside; HexPP, hexaprenyl diphosphate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Ogura, K., and Koyama, T. (1997) in Dynamic Aspects of Natural Products Chemistry: Molecular Biological Approaches (Ogura, K., and Sankawa, U., eds), pp. 1-23, Kodansha Ltd., Tokyo
  2. Ashby, M. N., and Edwards, P. A. (1990) J. Biol. Chem. 265, 13157-13164[Abstract/Free Full Text]
  3. Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J. Biochem. (Tokyo) 113, 355-363[Abstract]
  4. Chen, A., Kroon, P. A., and Poulter, C. D. (1994) Protein Sci. 3, 600-607[Abstract/Free Full Text]
  5. Tarshis, L. C., Yan, M., Poulter, C. D., and Sacchettini, J. C. (1994) Biochemistry 33, 10871-10877[Medline] [Order article via Infotrieve]
  6. Marrero, P. F., Poulter, C. D., and Edwards, P. A. (1992) J. Biol. Chem. 267, 21873-21878[Abstract/Free Full Text]
  7. Joly, A., and Edwards, P. A. (1993) J. Biol. Chem. 268, 26983-26989[Abstract/Free Full Text]
  8. Koyama, T., Saito, K., Ogura, K., Obata, S., and Takeshita, A. (1994) Can. J. Chem. 72, 75-79
  9. Song, L., and Poulter, C. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3044-3048[Abstract]
  10. Koyama, T., Obata, S., Saito, K., Takeshita-Koike, A., and Ogura, K. (1994) Biochemistry 33, 12644-12648[Medline] [Order article via Infotrieve]
  11. Koyama, T., Tajima, M., Nishino, T., and Ogura, K. (1995) Biochem. Biophys. Res. Commun. 212, 681-686[CrossRef][Medline] [Order article via Infotrieve]
  12. Koyama, T., Tajima, M., Sano, H., Doi, T., Koike-Takeshita, A., Obata, S., Nishino, T., and Ogura, K. (1996) Biochemistry 35, 9533-9538[CrossRef][Medline] [Order article via Infotrieve]
  13. Ohnuma, S., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095[Abstract/Free Full Text]
  14. Ohnuma, S., Narita, K., Nakazawa, T., Ishida, C., Takeuchi, Y., Ohto, C., and Nishino, T. (1996) J. Biol. Chem. 271, 30748-30754[Abstract/Free Full Text]
  15. Tarshis, L. C., Proteau, P. J., Kellogg, B. A., Sacchettini, J. C., and Poulter, C. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15018-15023[Abstract/Free Full Text]
  16. Ohnuma, S., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996) J. Biol. Chem. 271, 18831-18837[Abstract/Free Full Text]
  17. Ohnuma, S., Hirooka, K., Ohto, C., and Nishino, T. (1997) J. Biol. Chem. 272, 5192-5198[Abstract/Free Full Text]
  18. Allen, C. M., Keenan, M. V., and Sack, J. (1976) Arch. Biochem. Biophys. 175, 236-248[Medline] [Order article via Infotrieve]
  19. Muth, J. D., and Allen, C. M. (1984) Arch. Biochem. Biophys. 230, 49-60[Medline] [Order article via Infotrieve]
  20. Davisson, V. J., Woodside, A. B., Neal, T. R., Stremler, K. E., Muehlbacher, M., and Poulter, C. D. (1986) J. Org. Chem. 51, 4768-4779
  21. Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Bacteriol. 180, 1578-1581[Abstract/Free Full Text]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Raetz, C. R. H. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2274-2278[Abstract]
  24. Saito, H., and Miura, K. (1963) Biochim. Biophys. Acta 72, 619-629[CrossRef]
  25. Fujii, H., Koyama, T., and Ogura, K. (1982) Biochim. Biophys. Acta 712, 716-718[Medline] [Order article via Infotrieve]
  26. Fujii, H., Koyama, T., and Ogura, K. (1985) Methods Enzymol. 110, 192-198[Medline] [Order article via Infotrieve]
  27. Koyama, T., Yoshida, I., and Ogura, K. (1988) J. Biochem. (Tokyo) 103, 867-871[Abstract]
  28. Fujii, H., Koyama, T., and Ogura, K. (1982) J. Biol. Chem. 257, 14610-14612[Abstract/Free Full Text]
  29. Fujisaki, S., Hara, H., Nishimura, Y., Horiuchi, K., and Nishino, T. (1990) J. Biochem. (Tokyo) 108, 995-1000[Abstract]
  30. Asai, K., Fujisaki, S., Nishimura, Y., Nishino, T., Okada, K., Nakagawa, T., Kawamukai, M., and Matsuda, H. (1994) Biochem. Biophys. Res. Commun. 202, 340-345[CrossRef][Medline] [Order article via Infotrieve]
  31. Takatsuji, H., Nishino, T., Miki, I., and Katsuki, H. (1983) Biochem. Biophys. Res. Commun. 110, 187-193[Medline] [Order article via Infotrieve]
  32. Fujisaki, S., Nishino, T., Izui, K., and Katsuki, H. (1984) Biochem. Int. 8, 779-785[Medline] [Order article via Infotrieve]
  33. Ito, M., Kobayashi, M., Koyama, T., and Ogura, K. (1987) Biochemistry 26, 4745-4750[Medline] [Order article via Infotrieve]


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