Purification of solanesyl-diphosphate synthase from Micrococcus luteus. A new class of prenyltransferase.

The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography. By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa. In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate. HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner. Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis.

neither of which has any catalytic activity. It has been thought that the unique properties of these enzymes can account for their abilities to catalyze the synthesis of water-insoluble material from soluble substrates without aid of detergent molecules (28).
By analogy, it was expected that prenyltransferases responsible for the synthesis of (E)-polyprenyl diphosphates with chain lengths longer than CS5 would have properties similar to those of hexaprenyl-and heptaprenyl-diphosphate synthases. This seemed likely in view of the fact that solanesyl (all-E-nonapreny1)-diphosphate synthase (SPP' synthase) partially purified from M. luteus, unlike undecaprenyl-diphosphate synthase, does not require phospholipid or detergent. It has also been known that the partially purified enzyme catalyzes the synthesis of all-E-C40-prenyl diphosphate as well as all-E-C16-prenyl diphosphate under normal conditions (26) and that the chain lengths of products vary within the longest length of C45 depending on the concentration of M e ions in the reaction (27). However, it remained unclear whether the variability of the product chain length is a nature of a single enzyme, because further purification of the enzyme was difficult since the specific activity was apt to decrease during the course of purification.
The present studies were undertaken in order to clarify this point by purifying the enzyme to homogeneity as well as to learn whether the unique two-dissociable component system is common to all-E-polyprenyl-diphosphate synthases. This paper describes the achievement of purification of SPP synthase and the finding that this enzyme is quite different from hexaprenyl-and heptaprenyl-diphosphate synthases in terms of the constitution of subunits and the mode of activation.
from Whatman Chemical Separation, Inc. All other chemicals were of analytical grade. SPP Synthase Assay-The enzyme activity was assayed as usual by measuring the amount of incorporation of [1-14C]IPP into butanolextractable polyprenyl diphosphates. In a standard experiment the assay mixture contained, in a final volume of 1.0 ml, 20 *mol of Tris-HC1 buffer (pH 7.7), 5 pmol of MgC12, 25 nmol of FPP, 25 nmol of [l-"C]IPP (37 GBq/mol), S P P synthase, and HMF, the preparations of which are described later. The mixture was incubated a t 37 "C for 1 h, and the reaction was stopped by chilling the reaction mixture in a n ice bath. The mixture was shaken with 3.0 ml of butanol that had been saturated with H20. The butanol phase was back-washed with water, and the radioactivity in the butanol phase was determined.
Analysis of Products-The polyprenyl diphosphates produced by the enzymatic reactions were treated with acid phosphatase according to the method of Fujii et al. (30). The hydrolysates were extracted with pentane, and the pentane-soluble products were analyzed by reversed phase LKC-18 thin layer chromatography and silica gel chromatography developed with a solvent system of acetone/HzO (19:l) and benzene/ethyl acetate (9:1), respectively. The positions of authentic standards were visualized with iodine vapor. Distribution of radioactivity was detected by scanning the plate with an Aloka radiochromatoscanner.
Preparation of HMF and Purification of SPP Synthase-Freezedried cells (30 g) were suspended in 450 ml of 50 mM Tris-HC1 buffer (pH 7.7) containing 1 mM EDTA (buffer A). Lysozyme (150 mg) was added to the cell suspension, and the mixture was stirred for 40 min at room temperature. The lysate was then treated with deoxyribonuclease (30 mg) at 0 "C for 1 h and centrifuged a t 100,000 X g for 1 h. The supernatant fraction was chromatographed on a DEAE-Toyopearl 650M column (7 X 25 cm) equilibrated with buffer A.
The SPP synthase fractions (fraction numbers 102-110) were pooled and concentrated to 20 ml by ultrafiltration with a YM 10 membrane (Amicon Co. Ltd.). The concentrated enzyme solution was applied to a Sephadex G-100 column (4 X 72 cm) equilibrated with buffer A.
Elution was performed with buffer A (see Fig. 6A). The SPP synthase activity was stimulated by fraction I (fraction numbers 27-37), which is designated as HMF. The SPP synthase activity was found in fraction I1 (fraction numbers 39-46) with a reduced yield (see Fig.   6B). The enzyme fraction was chromatographed on a Mono Q column (10 X 100 mm) equilibrated with buffer A. Elution was performed with a programmed gradient of 0-400 mM of NaCl (Fig. 2). The SPP synthase fractions (retention time, 37-39 min) were combined and then chromatographed on a Butyl-Toyopearl 650M column (1.6 X 10 cm) equilibrated with buffer A containing ammonium sulfate a t 40% saturation. Elution was performed with a decreasing linear gradient The supernatant fraction was chromatographed on a DEAE-Toyopearl650M column (7 X 25 cm) equilibrated with buffer A. Elution was performed with a gradient of 0-850 mM NaCl (20 ml/fraction). Each fraction was assayed in the absence of HMF as described under "Materials and Methods." Fractions 88-96 included (all-E)-geranylgeranyl-diphosphate synthase. Fractions 102-110 included SPP synthase. Fractions 95-98 included undecaprenyl-diphosphate synthase, the activity of which was not detected without a supplement of detergent. 0, enzyme activity with FPP and [1-14C]IPP; ---, absorbance a t 280 nm; 0, conductivity. fractions containing ammonium sulfate at 20% saturation were chromatographed on a Butyl-Toyopearl 650M column (1.6 X 10 cm) equilibrated with 50 mM Tris-HC1 buffer, pH 7.7, containing 1 mM EDTA and ammonium sulfate a t 40% saturation. Elution was carried out with a linear gradient of decreasing amount of ammonium sulfate in 50 min a t a flow rate of 2 ml/min. Each fraction was assayed in the presence of HMF as described under "Materials and Methods." 0 , S P P synthase activity; ---, absorbance at 280 nm: --, concentration of (NH&SO4. from 40 to 0% saturation of ammonium sulfate in buffer A (Fig. 3).
The SPP synthase fractions (retention time, 59-61 min) were pooled and concentrated by ultrafiltration with a Centricon-10 membrane filter (Amicon Co. Ltd.). The concentrated solution was applied to a Superose 12 column (1.0 x 30 cm) equilibrated with buffer A (Fig. 4).
The SPP synthase fractions (retention time, 25-28 min) were pooled and concentrated by ultrafiltration with a Centricon-10 membrane filter. The concentrated solution was loaded on a native polyacrylamide gel (pH 8.9, 7.5%), and the gel electrophoresis was run by standard methods (31). Part of the protein was stained with Coomassie Blue or silver (Bio-Rad). The protein band in the gel was sliced in 5-mm widths, and the protein was eluted from the sliced gel with an electrophoretic concentrator (LKB Extraphor). The eluted protein was assayed for S P P synthase activity (Fig. 5A). The nativepolyacrylamide gel electrophoresis (PAGE)-purified SPP synthase was shown to he homogeneous by both SDS-PAGE (IO%, see Fig. 7) run by standard methods (32) with silver-staining and native PAGE (Fig. 5B). The protein assay kit (Bio-Rad) with lipid-free bovine serum albumin was used as a protein standard for all protein concentration determinations. The HMF fraction resulting from Sephadex G-100 chromatography and the SPP synthase fraction from Mono Q chromatography were used in most of the experiments for their characterization.  Distoncelcm FIG. 5. Native PAGE of the Superose 12-purified SPP synthase. A, activity of S P P synthase recovered from native PAGE (7.5%). The assay methods were described under "Materials and Methods." B, the silver-stained gel of native PAGE. The native PAGE-purified SPP synthase was re-electrophoresed under the same conditions and silver-stained.

RESULTS
Preparation of HMF-When the DEAE-Toyopearl-purified SPP synthase ( Fig. 1) was chromatographed on a Sephadex G-100 column (Fig. 6A), the enzymatic activity decreased to less than 10% of that before the chromatography. It has been shown that some bacterial prenyltransferases have multicomponent systems; hexaprenyl-diphosphate synthase (22,23) from M. luteus B-P 26 or heptaprenyl-diphosphate synthase (24, 25) from B. sdtilis is composed of two easily dissociable and nonidentical subunits. On the other hand, undecaprenyl-diphosphate synthase, which is composed of nondissociable and identical subunits, requires some lipids for its catalytic activity. Recently, rubber synthase (33-36) from Hevea brasiliensis has been found to be composed of two nonidentical subunits, one having a FPP synthase activity and the other an elongation factor. Assuming that a factor or factors which affect the activity of SPP synthase were deprived during the Sephadex G-100 chromatography, we first searched for such a factor in the fractions obtained in the gel filtration. As shown in Fig, 6B, a high molecular mass fraction (I, fraction numbers 27-37) stimulated the SPP synthase activity. This fraction, designated as HMF, was used in the following experiments.
Purification of SPP Synthase-The purification procedure of M. luteus SPP synthase included two kinds of gel filtra- tions, two kinds of ion exchange chromatographies, hydrophobic chromatography and native PAGE ( Table I). The assay for enzyme activity was carried out with a supplement of HMF. The native PAGE-purified SPP synthase gave on SDS-PAGE a single protein band corresponding to a molecular mass of 34 kDa (Fig. 7). Since the molecular mass of SPP synthase was 74-78 kDa as estimated by either Superose 12 ( Fig. 4) or Sephadex G-100 chromatography (Fig. 6A), the enzyme seems to be a dimer with a monomeric molecular mass of 34 kDa. The pure SPP synthase was catalytically active even in the absence of HMF, but it was activated by HMF more markedly than was a partially purified preparation of SPP synthase. Product Analysis-The products formed by the reaction of the native PAGE-purified SPP synthase in the presence or absence of HMF were analyzed. As the reaction products were diphosphate esters, they were hydrolyzed with acid phosphatase as usual (30) and the hydrolysates were analyzed by means of reversed phase and silica gel thin layer chromatographies. As shown in Fig. 8, in the case of the reaction of pure SPP synthase without addition of HMF, one of the major radioactive products migrated to the region where authentic (all-E)-nonaprenol (solanesol) migrated. Another radioactive product migrated faster than authentic solanesol and slower than authentic (22,62,102,142,182,22E,26E)-octaprenol. In view of the fact that an (all-E)-polyprenol migrates slower than its 2,E-mixed isomer (24), these products seems to be (all-E)-nonaprenol (C4s) and (all-E)-octaprenol (c40). These products co-migrated with the authentic C40 and C4s prenols on silica gel thin layer chromatography respectively (data not shown). The distribution of these products is similar to that observed with partially purified SPP synthase (26). The distribution pattern of the products formed in the absence of HMF was almost the same as that of the products formed in its presence.
High Molecular Fraction Which Activates SPP Synthase-HMF was found to be precipitable with ammonium sulfate at 50% saturation (data not shown). In order to see whether or not lipids are involved in HMF, we carried out lipid extraction experiments according to the Bligh-Dyer method (37). As shown in Table 11, the lipid extracts did not stimulate the activity of SPP synthase, but the water-soluble fraction did stimulate the enzyme activity, although the effect was slightly lower than that of the untreated HMF. Anthrone reagent was used for detection of any saccharides in HMF, but no saccha- ' Precise amounts of SPP synthase could not be determined by the assay method described under "Materials and Methods," because these fractions contained other prenyltransferases (farnesyl-, geranylgeranyl-, and undecaprenyl-diphosphate synthases) and phosphatase, which would interfere in the measurement of SPP synthase activity.
ND, not determined.

SDS-PAGE
-" --- rides were detected. By assaying the enzymatic activity using HMF instead of FPP, we also ascertained that HMF did not contain any endogenous allylic diphosphates that would act as primer substrates. From these facts, the stimulation seems to be attributable to protein in HMF.
The effect of HMF on SPP synthase activity is shown in Fig. 9A. As the amount of HMF was increased, the enzyme activity increased up to approximately 3-fold. Bovine serum albumin (BSA) also showed a similar stimulative effect (Fig.  9 B ) .
SPP synthase itself was labile against heat treatment, but HMF was stable even when treated at 55 "C for 10 min (Fig.   1OA). We therefore examined whether or not the activation by HMF is a result of stabilization of SPP synthase against heat treatment. The addition of HMF, however, did not stabilize the enzyme (Fig. 10B).
The amount of the products formed by the enzyme reaction was determined as a function of incubation period in the presence or absence of HMF (Fig. 1lA). In these experiments, 40 pg of the Mono Q-purified SPP synthase and 750 pg of HMF protein were employed. In the presence of HMF, the reaction proceeded almost linearly for 4 h. In its absence, however, the reaction reached a plateau within 1 h. Namely,

Effect of the lipid fraction and water-soluble fraction separated from HMF
The lipid fraction and the water-soluble fraction were prepared from HMF according to the method of Bligh and Dyer (38). Solvents of these fractions were evaporated in uacuo, and the residues were dissolved in water. The activity was assayed under the standard conditions described under "Materials and Methods" except that the h i d fraction and the water-soluble fraction were added as indicated. Additive Lipid fractionb + water soluble

5,078
fraction" a 400 pg of protein was used. *The amount of the lipid fraction was adjusted to a level similar to that of the untreated HMF fraction.  Sephadex G-100-purified HMF (0) and the Mono Q-purified SPP synthase (0) were incubated at 55 "C without substrates for the indicated period, and then assayed for their remaining activity with a supplement of untreated SPP synthase and untreated HMF, respectively, under the standard conditions described under "Materials and Methods." B, the Mono Q-purified SPP synthase was heated in the presence of the Sephadex G-100-purified HMF at 55 "C for the indicated period, and then assayed for the restorative activity for SPP synthase (0). The calculated values (0) were obtained from the data shown in Fig. 3A by means of multiplication of heat stabilities of SPP synthase and HMF. the observed stimulation of the enzyme activity depended markedly on the incubation period; the activations were about 3-and 10-fold after 1 and 4 h, respectively. To examine whether the enzyme was denatured after the incubation, we carried out experiments as follows. After the enzymatic reaction was carried out in the absence of HMF for indicated periods, HMF was added to the reaction mixture, and then the mixture was incubated again at 37 "C for 1 h (Fig. 11B).
The enzyme was still active, and the reaction proceeded again.
Kinetic parameters of SPP synthase reaction in the presence or absence of HMF were determined (Table 111)  its absence in the 20-min incubation. However, the K , values estimated in the absence of HMF were both similar to those in its presence. Thus, HMF seems to stimulate the SPP synthase activity by increasing the initial rate of the reaction as well as maintaining the turnover of reaction for several hours. It has been reported that the long chain polyprenyl diphosphates produced by a partially purified enzyme from M. luteus are eluted in the Vo region of Sephadex G-25 (38). In order to examine whether HMF shows some specific interaction with the substrates or products, we carried out binding experiments by means of Superose 12 chromatography, using a mixture of [I4C]octaprenyl diphosphate and ['4C]solanesyl diphosphate synthesized from [1-'4C]IPP and FPP by SPP synthase in the presence or absence of HMF. When the mixture resulting from the reaction carried out without HMF was chromatographed on a Superose 12 column, two peaks of radioactivity emerged (Fig. 124). Peak 11, which emerged in the Vt region, corresponded to recovered IPP, while Peak I, which emerged near the elution point of SPP synthase, corresponded to the products including solanesyl and octaprenyl diphosphates. In the case of the reaction mixture in the presence of HMF, two peaks of radioactivity also emerged from the column (Fig. 12B). Peak I1 had an elution volume similar to that observed in the former column. The reaction products, solanesyl and octaprenyl diphosphates, emerged together with HMF at the Vo region, forming Peak 111. Both [14C]IPP and [14C]FPP were eluted in the Vt region of the column without regard to the presence or absence of HMF (data not shown).
In order to investigate the state of the polyprenyl products in Peak 111, similar binding experiments were carried out employing BSA or bacitracin instead of HMF. It has been reported that the former binds to some hydrophobic molecules nonspecifically and that the latter exhibits high affinity for polyprenyl diphosphate (39). Both BSA (Fig. 9B) and bacitracin (data not shown) also stimulated SPP synthase activity, and the extent of the stimulation depended on their concentration. When a reaction mixture containing BSA was incubated and then chromatographed on a Superose 12 column, two radioactivity peaks, Peaks IV and 11, emerged from the column (Fig. 12C). The elution volume of Peak I1 was similar again to that observed in Fig. 12, A and B while Peak IV, which was proved to contain the enzymatic products, was eluted together with BSA. In the presence of bacitracin, the reaction mixture gave only a single peak of radioactivity in the Vt region of Superose 12 chromatography (data not shown), and it was found that the fraction corresponding to this peak contained ['4C]IPP as well as the enzymatic products [14C]SPP, [ 14C]octaprenyl diphosphate, and bacitracin. These results indicate that not only HMF but also materials that can bind to the polyprenyl products stimulate the SPP synthase activity.

DISCUSSION
The discovery of the HMF, which markedly stimulates SPP synthase activity, facilitated the purification of SPP synthase from M. luteus, and the enzyme was purified to homogeneity. TO our knowledge, this is the first report of purification to electrophoretic homogeneity of a prenyltransferase that produces the precursor of quinone side chain. This enzyme is a homodimer like other prenyltransferases that have been purified so far (12, 15, 40, 41). In the presence or absence of HMF, the purified enzyme catalyzes the synthesis of both (all-E)-octaprenyl diphosphate and (all-E)-nonaprenyl (solanesyl) diphosphate, which are the precursors of the side chains of menaquinones 8 and 9, occurring in this bacterium (42). In this respect this enzyme is different from other polyprenyl-diphosphate synthases. Undecaprenyl-diphosphate synthase, which catalyzes cis-chain elongation to produce 2,E-mixed C55-prenyl diphosphate, essentially requires phospholipids or some detergent for its catalytic activity (13-18). Hexaprenyl-diphosphate synthase (22,23)  subtilis is composed of two dissimilar subunits, each of which has no catalytic activity at all.
Recently, rubber synthase (33-36) from H. brasiliensis has been shown to have two different prenyltransferase activities.
In the presence of "rubber elongation factor" it catalyzes the addition of multiple cis-isoprene units to rubber molecules, while in the absence of rubber elongation factor this enzyme shows FPP synthase activity, that is, the addition of two trans-isoprene units to dimethylallyl diphosphate.

Solanesyl Diphosphate Synthase
On the other hand, the homogeneously purified protein in this study shows SPP synthase activity by itself. Investigation of the cause for the decrease in specific activity during its purification led us to the finding that SPP synthase activity was stimulated by HMF. The stimulation depends on the concentration of HMF, but it is not an absolute requirement for catalytic activity. In contrast to rubber synthase, the function of which is dramatically changed by rubber elongation factor, the function of SPP synthase was not altered by HMF at all. The chain length distribution of the products of the purified SPP synthase was found to change dependent on Mg2+ concentration (data not shown) as observed previously (27) with a partially purified enzyme.
The fact that HMF is heat-stable is reminiscent of component A of hexaprenyl-diphosphate synthase (22) or component I of heptaprenyl-diphosphate synthase (25), but HMF cannot substitute for any of these components (data not shown). HMF does not affect the heat stability of SPP synthase in the absence or presence of substrates (Figs. 5B and  6 B ) . Many carrier proteins or binding proteins which directly participate in the bioconversion of hydrophobic molecules are known. Some bind to substrates and others to products. For example, sterol carrier protein-2 (43, 44) participates in the conversion of squalene to lanosterol by liver microsomal enzyme, and fatty acid-binding protein (45) exhibits a high affinity for fatty acids and their CoA esters and may participate in their intracellular transport. These proteins share a functional similarity by allowing insoluble molecules to move through and interact with components in the aqueous environment.
HMF not only increases the velocity of SPP synthase reaction depending on its concentration but also extends the linearity of the reaction. In the case of FPP synthase reaction, deprivation of the product FPP, which is a potent inhibitor (46), has been reported to be the rate-limiting step (47). As the long prenyl chain of the product of a polyprenyl-diphosphate synthase would have much hydrophobic interaction with the active site of the enzyme, it should have a mechanism to deprive the enzyme of its product to maintain the turnover of catalysis. It is reasonable to assume that HMF contains a factor that plays a functional role in removal of hydrophobic products from the active site of enzyme.
Several lines of evidence indicated that HMF contains a factor which binds to polyprenyl diphosphzkes synthesized by SPP synthase, but not to either of the substrates, IPP and FPP. It was also shown that both BSA and bacitracin could bind to SPP and stimulate the catalytic activity of SPP synthase. The former is known to bind to some hydrophobic molecules nonspecifically, and the latter to exhibit high affinity for polyprenyl diphosphates (39). These results suggest that the factor (HMF) stimulates the enzyme activity by removing SPP from the enzyme. Trypsin treatment of HMF did not result in a complete loss of activity (data not shown), but this may indicate that the lysate of this factor is also active rather than that the factor is not protein.
In order to see whether the stimulation effect of HMF is specific for SPP synthase from M. luteus, we examined the effect of HMF on other prenyltransferases from various organisms. As a result, HMF did not stimulate any of undecaprenyl-diphosphate synthase from B. subtilk, hexaprenyldiphosphate synthase from M. luteus B-P 26, and FPP synthase from pig liver. However, it stimulated the octaprenyldiphosphate synthase from Escherichia coli K-12 and decaprenyl-diphosphate synthase from Paracoccus denitrificans (data not shown). Therefore HMF is commonly effective to bacterial prenyltransferases that catalyze the synthesis of all-E-polyprenyl diphosphates with chain lengths of C40, C45, and c 5 0 . These three all-E-polyprenyl-diphosphate synthases, also share a similar property in that they are stimulated by Tween 80, which does not affect hexaprenyl-or heptaprenyl-diphosphate synthase (data not shown). Therefore, they should be assigned to a new class different from that for hexaprenyland heptaprenyl-diphosphate synthases. The involvement of HMF must be essential because of the molecular properties of these particular products that each consist of a polyprenyl chain of Cd0, C45, or C5, length and a diphosphate moiety.
It is surprising that the properties of M. luteus SPP synthase reaction are quite different from those of hexaprenyldiphosphate synthase from M. luteus B-P 26. Although M. luteus B-P 26 has been classified in the same Micrococcus subgroup as the strain of ATCC (48), the characteristics of these two strains are quite different from each other in that the former is nonpigmented and is resistant to lysozyme treatment.