Reverse Gyrase from Methanopyrus kandleri

Reverse gyrases are ATP-dependent type I 5′-topoisomerases that positively supercoil DNA. Reverse gyrase from Methanopyrus kandleri is unique as the first heterodimeric type I 5′-topoisomerase described, consisting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) and a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA). Here we report the reconstitution of active reverse gyrase from the two recombinant proteins overexpressed in Escherichia coli. Both proteins have been purified by column chromatography to >90% homogeneity. RgyB has a DNA-dependent ATPase activity at high temperature (80 °C) and is independent of the presence of RgyA. RgyA alone has no detectable activity. The addition of RgyA to RgyB reconstitutes positive supercoiling activity, but the RgyB and RgyA subunits form a stable heterodimer only after being heated together. This is the first case in which it has been possible to reconstitute an active heterodimeric enzyme of a hyperthermophilic prokaryote from recombinant proteins. Reverse gyrases are ATP-dependent type I 5′-topoisomerases that positively supercoil DNA. Reverse gyrase from Methanopyrus kandleri is unique as the first heterodimeric type I 5′-topoisomerase described, consisting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) and a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA). Here we report the reconstitution of active reverse gyrase from the two recombinant proteins overexpressed in Escherichia coli. Both proteins have been purified by column chromatography to >90% homogeneity. RgyB has a DNA-dependent ATPase activity at high temperature (80 °C) and is independent of the presence of RgyA. RgyA alone has no detectable activity. The addition of RgyA to RgyB reconstitutes positive supercoiling activity, but the RgyB and RgyA subunits form a stable heterodimer only after being heated together. This is the first case in which it has been possible to reconstitute an active heterodimeric enzyme of a hyperthermophilic prokaryote from recombinant proteins. In the 1980s organisms were discovered that thrive at temperatures >100 °C. These hyperthermophiles have provided a host of enzymes for scientific study and potential industrial application (1Adams M.W.W. Perler F.B. Kelly R.M. Bio/Technology. 1995; 13: 662-668Google Scholar). However, many archaea fail to grow in the laboratory (2Huber R. Burggraf S. Mayer T. Barns S.M. Rossnagel P. Stetter K.O. Nature. 1995; 376: 57-58Google Scholar), and the majority that can be grown do so inefficiently (3Robb F.T. Place A.R. Archaea: A Laboratory Manual, Thermophiles. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995Google Scholar). To fully realize the potential benefits of these enzymes and to make them easily accessible for study in the laboratory, it is advantageous to use recombinant DNA technology to express hyperthermophilic proteins in heterologous hosts and then recover active enzymes (1Adams M.W.W. Perler F.B. Kelly R.M. Bio/Technology. 1995; 13: 662-668Google Scholar, 4Adams M.W.W. Kelly R.M. Chem. Eng. News. 1995; : 32-42Google Scholar). Although many active thermostable enzymes have been recovered after expression in Escherichia coli, until now it had not been possible to reconstitute an active heteromultimeric hyperthermophilic enzyme from recombinant proteins (1Adams M.W.W. Perler F.B. Kelly R.M. Bio/Technology. 1995; 13: 662-668Google Scholar). Reverse DNA gyrases, a novel class of type I 5′-topoisomerases, were discovered in hyperthermophiles (5Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Google Scholar, 6Nakasu S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Google Scholar, 7Slesarev A.I. Eur. J. Biochem. 1988; 173: 395-399Google Scholar, 8Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Google Scholar, 9Kovalsky O.I. Kozyavkin S.A. Slesarev A.I. Nucleic Acids Res. 1990; 18: 2801-2805Google Scholar, 10Confalonieri F. Elie C. Nadal M. Bouthier de la Tour C.B. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Google Scholar, 11Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar). These enzymes, which positively supercoil DNA in the presence of ATP or dATP, have been found in all major branches of hyperthermophilic archaea as well as in hyperthermophilic eubacteria (12Bouthier de la Tour C. Portemer C. Nadal M. Stetter K.O. Forterre P. Duguet M. J. Bacteriol. 1990; 172: 6803-6808Google Scholar, 13Bouthier de la Tour C. Portemer C. Huber R. Forterre P. Duguet M. J. Bacteriol. 1991; 173: 3921-3923Google Scholar). Reverse gyrase is thus considered a molecular marker for hyperthermophilic prokaryotes (14Forterre P. Cell. 1996; 85: 789-792Google Scholar). Although the physiological role of the activity remains obscure, it has been postulated that positive supercoiling helps to stabilize the DNA duplex against denaturation at the extreme temperatures where these organisms grow. In the eubacteria and the crenarchaeota branch of the archaea, reverse gyrases isolated to date are large monomeric proteins (12Bouthier de la Tour C. Portemer C. Nadal M. Stetter K.O. Forterre P. Duguet M. J. Bacteriol. 1990; 172: 6803-6808Google Scholar, 13Bouthier de la Tour C. Portemer C. Huber R. Forterre P. Duguet M. J. Bacteriol. 1991; 173: 3921-3923Google Scholar). Sequence analysis of reverse gyrase fromSulfolobus acidocaldarius revealed that the type I 5′-topoisomerase domain was fused to a domain reminiscent of a nucleic acid helicase (10Confalonieri F. Elie C. Nadal M. Bouthier de la Tour C.B. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Google Scholar). Helicases are nucleic acid-dependent ATPases as is reverse gyrase (15Shibata T. Nakasu S. Kumiko Y. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Google Scholar). However, it is important to note that this domain of reverse gyrase may not function as a helicase during the enzymatic reaction and the conserved amino acid motifs in this enzyme may serve another function. Recently, we described a reverse gyrase from Methanopyrus kandleri, the first type I 5′-topoisomerase isolated from the euryarchaeota branch of the archaea (16Rivera M.C. Lake J.C. Int. J. Syst. Bacteriol. 1996; 46: 348-351Google Scholar, 17Kozyavkin S.A. Krah R. Gellert M. Stetter K.O. Lake J.A. Slesarev A.I. J. Biol. Chem. 1994; 269: 11081-11089Google Scholar, 18Krah E.R. Kozyavkin S.A. Slesarev A.I. Gellert M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 106-110Google Scholar). Unlike any other type I topoisomerase, this enzyme is a heterodimer with subunits of 43 kDa (RgyA) and 138 kDa (RgyB). It was demonstrated that RgyA forms the covalent link with DNA during the strand cleavage-religation reactions, whereas RgyB is involved in the hydrolysis of ATP (17Kozyavkin S.A. Krah R. Gellert M. Stetter K.O. Lake J.A. Slesarev A.I. J. Biol. Chem. 1994; 269: 11081-11089Google Scholar). We expected the two protomers of M. kandleri reverse gyrase to consist of the conserved domains found in S. acidocaldarius reverse gyrase but as separate proteins. However, the cloning and sequence analysis of the genes encoding the two subunits of M. kandleri reverse gyrase revealed the unexpected result that the universally conserved type I 5′-topoisomerase domain was not continuous but instead was shared between the two subunits. To study the activities of the enzyme and its subunits in greater detail, it is useful to generate larger amounts of protein than can conveniently be made in M. kandleri. Here, we report growth conditions for the recovery of soluble recombinant RgyA and RgyB fromE. coli and the reconstitution of active, stable reverse gyrase from the purified proteins. Restriction enzymes, T4 DNA ligase, Vent DNA polymerase, calf alkaline phosphatase, and deoxyribonucleotides were purchased from New England Biolabs Inc. and used as described by the manufacturer. pET-3 and pET-16b protein expression vectors were from Novagen. Polyethyleneimine (Sigma) was used as described (19Burgess R.R. Methods Enzymol. 1991; 208: 3-10Google Scholar). Isopropyl-1-thio-β-d-galactopyranoside was from Gold Biotechnology Inc. HiTrap chelating and heparin columns and a Sephadex S200HR 10/30 sizing column were from Pharmacia Biotech Inc. High resolution hydroxylapatite (HTH) 1The abbreviations used are: HTH, hydroxylapatite; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. was from Calbiochem. Phosphocellulose P-11 was from Whatman. Both L-broth and SuperbrothMDRV were purchased from Advanced Biotechnologies Inc. Column chromatography was performed using a Pharmacia fast protein liquid chromatography apparatus. All chromatography steps were performed at 4 °C except for the elution of the HTH column of RgyA, which was done at room temperature. Analytical gel filtration was performed using a Pharmacia Smart system with a Superdex 200 3.2/30 column that was standardized using the Pharmacia high and low molecular weight gel filtration kits. For electrophoretic analysis, protein samples were diluted as indicated in the figure legends, mixed 1:1 with Tris/glycine SDS sample buffer (Novex), and separated on 4–24% gradient SDS-PAGE gels (Novex). Proteins were visualized by staining with Coomassie Brilliant Blue (0.25% in 45% methanol, 10% glacial acetic acid) followed by destaining with 10% glacial acetic acid. Protein purity was determined by scanning gels of protein samples with a Microtek Scanmaker II and creating a computer file with Adobe Photoshop and then analyzing band intensity with the NIH Image computer program. RgyB was first preheated in its storage buffer for 5 min at 80 °C. Then 0.1 μg of the protein was combined with 0.1 μg of supercoiled pBR322 in a 20-μl volume of reaction buffer (50 mm Tris, pH 8, 200 mm NaCl, 10 mm MgCl2, 1 mm DTT, 10% w/v glycerol) containing 50 μm [γ-32P]ATP (600 mCi/mmol, DuPont NEN). Samples were incubated at the temperatures and times indicated in the figure legends, and the reactions were terminated by rapid cooling and dilution into 100 mm EDTA, pH 8. A 1-μl aliquot of each sample was spotted on a 20 × 20-cm polyethyleneimine cellulose plastic plate (Alltech Associates Inc.), dried, and developed by ascending chromatography for 2 h with 0.75m KH2PO4. Radioactivity was quantitated with a PhosphorImager (Molecular Dynamics, Inc.). To reconstitute positive supercoiling activity, we mixed amounts of RgyA and RgyB as indicated in their storage buffer (50 mm Tris, pH 8, 1 m NaCl, 1 mm EDTA, 10% glycerol) and heated for 5 min at 80 °C. For tests of positive supercoiling activity, 1 μl of enzyme (0.1 μg of either the A subunit, the B subunit, or the reconstituted reverse gyrase) was mixed with 0.1 μg of pBR322 DNA (either relaxed or negatively supercoiled, as specified) in 20 μl of reaction buffer (50 mm Tris, pH 8, 200 mm NaCl, 5% glycerol, 10 mm MgCl2) and incubated at 80 °C for 15 min. The reactions were terminated by rapidly cooling to 0 °C and adding 0.2 volume of 5% SDS, 50 mm EDTA, 50% glycerol, 0.5% bromphenol blue. The topoisomerization products were analyzed by electrophoresis in a 1.2% agarose gel in the presence or absence of chloroquine at 3 V/cm for 12 h. RgyA and RgyB were produced separately in E. coli using the pET expression system of Studier et al. (20Studier F.W. Rosenber A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Google Scholar). ThergyB coding sequence was amplified by polymerase chain reaction from a recombinant λ phage carrying the entire gene and cloned into the NdeI-BamHI site of pET-16b, placing a 22-amino acid leader containing a poly(His) sequence onto the amino terminus of the protein. rgyA was amplified by polymerase chain reaction from a recombinant λ phage that carried the full-length gene. The first codon of the open reading frame was changed from TTG to ATG, and a second alternative translation start site in a different reading frame adjacent to the first codon was removed by making a silent mutation in a Asn codon (changing AAT to AAC).rgyA was cloned into the NdeI-BamHI site of pET3. Four liters of Superbroth were inoculated with BL21::λDE3 pLysE carrying the RgyA or RgyB expression plasmid. Cells were grown at 37 °C to anA600 of 0.8 with aeration, then isopropyl-1-thio-β-d-galactopyranoside (final concentration, 400 μm) was added, and the culture immediately shifted to 20 °C for protein expression. After 8 h the cells were harvested, yielding approximately 16 g of cells that were then resuspended in 40 ml of freezing buffer (50 mm Tris, pH 8, 10% sucrose, 2 mm EDTA, 100 mm NaCl), frozen in liquid nitrogen, and stored at −70 °C. The cell paste was thawed on ice, and an equal volume of freezing buffer containing lysozyme (0.2 μg/ml) was added. After stirring for 30 min at 4 °C, an equal volume of lysis buffer was added (50 mm Tris, pH 8, 2 mm DTT, 5 mm EDTA, 200 mm NaCl, 0.4% Brij 58) and stirred for 30 min followed by shearing of the cell lysate with a rotary homogenizer (Janke and Kunkel, Ultra-Turrax T25) to reduce viscosity. Cellular debris was removed by centrifugation at 32,000 × g for 30 min at 4 °C, after which a 0.07 volume of 5% polyethyleneimine chloride, pH 8, was slowly added to the cleared lysate and then stirred for 30 min. The resulting aggregate was collected by centrifugation and washed with 50 ml of buffer A (50 mm Tris, 2 mm EDTA, 2 mm DTT) containing 300 mm NaCl to remove loosely bound protein. Proteins were removed from the polyethyleneimine aggregate with 50 ml of buffer A containing 1 m NaCl and precipitated by the addition of solid ammonium sulfate to a concentration of 50% (w/v). At this point the purifications of RgyA and RgyB diverged. The RgyA ammonium sulfate pellet was resuspended in 25 ml of buffer A (with 1 m NaCl) and dialyzed overnight against 2 liters of Buffer A (with 300 mm NaCl) containing 10% glycerol. Under these conditions, RgyA preferentially precipitates out of solution. This precipitate was collected and then redissolved in 15 ml of buffer B (50 mmTris, pH 8, 1 m NaCl, 2 mm DTT, 10% glycerol) plus 10 mm potassium phosphate and 0.1 mm EDTA. The sample was pumped through a 6-ml phosphocellulose column directly onto a 2-ml HTH column that had been pre-equilibrated with buffer B with 10 mm potassium phosphate. The phosphocellulose column was removed, and RgyA was eluted from the HTH column with 20 column volumes of a linear 0.01–0.5 m potassium phosphate gradient, pH 7.4, in buffer B. Under these conditions, RgyA eluted as two separate peaks centered on fractions 7 and 13 (Fig.1). The protein in both peaks behaves identically in the assays shown below. The second peak contains more pure RgyA protein than peak 1. Fractions corresponding to peak 2 were pooled, dialyzed against 2 liters of storage buffer (buffer A plus 1 m NaCl, 10% and used in The fractions of peak 2 of protein that was >90% pure of reverse gyrase in a The ammonium RgyB was resuspended in 20 ml of buffer (50 mm Tris, pH 8, NaCl, 2 mm containing 10 mm and 0.1 mm EDTA, pumped through a phosphocellulose and directly onto a The phosphocellulose column was removed, and the column was washed with ml of buffer containing m followed by a gradient from to 2 m in buffer ml Fractions containing RgyB were and dialyzed overnight against 2 liters of buffer (50 mm Tris, pH 8, 100 0.1 mm EDTA, and 2 mm precipitate was that was removed by The sample was onto a HiTrap heparin column at 0.1 and washed with 25 ml of buffer and the protein was eluted with a linear gradient of m NaCl in buffer (50 ml Fractions in which the major protein was full-length RgyB were pooled, to 10 mm potassium and directly onto a 2-ml HTH and the sample was washed with 10 ml of Buffer (50 mm Tris, pH 8, 200 mm NaCl, 10% glycerol) plus 10 mm potassium phosphate. The protein was eluted with a linear gradient ml from 10 to mm potassium pH 7.4, in buffer E. Fractions in which the major protein was full-length RgyB were The sample was into separate and each aliquot was separated on a Superdex 10/30 sizing column pre-equilibrated with buffer A with 1 m NaCl and 10% w/v glycerol. Samples of full-length RgyB were pooled, yielding of protein RgyA protein was overexpressed in E. coli at 37 °C, but was by the protein at 20 °C. After of the cells and lysis as described approximately 50% of the overexpressed protein in the soluble of the cleared The of RgyA in the soluble the concentration of the lysis buffer was to 100 mm The of soluble RgyA may on the of E. coli. translation at a temperature may slowly to of to of other recombinant proteins from hyperthermophiles by growth at temperatures has been (1Adams M.W.W. Perler F.B. Kelly R.M. Bio/Technology. 1995; 13: 662-668Google Scholar). The of recombinant RgyB was by the protein at 20 °C not RgyB and translation during expression in E. a sample 2 However, by being in which fractions were during it was possible to a sample that was full-length a for RgyA and a for each subunit was purified to >90% as determined by scanning analysis of SDS-PAGE gels of each Although RgyA eluted as two separate peaks from an HTH column these two fractions had that were by In a supercoiling of 1 μl of each column of RgyA from the HTH column with 25 μg of purified RgyB revealed two peaks of enzymatic activity corresponding with the two peaks of RgyA protein that eluted from the RgyA samples from peak 1 and peak 2 both in the volume of an sizing a to form high molecular protein from either peak of RgyA can form a stable heterodimer with RgyB at high temperature We that these two peaks may two different of RgyA that have different for the HTH In the steps of the RgyA had a to precipitate out of at We used this to in the by the precipitate that during from NaCl to 300 mm NaCl This precipitate in containing 1 and not protein. After of the sample through a phosphocellulose RgyA in on into containing as as 100 mm We this of fractions is to polyethyleneimine in the sample that is removed by through the phosphocellulose column (19Burgess R.R. Methods Enzymol. 1991; 208: 3-10Google Scholar). all isolated reverse gyrases were enzymes (14Forterre P. Cell. 1996; 85: 789-792Google Scholar, T. Nakasu S. Kumiko Y. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Google Scholar), it had not been possible to the DNA-dependent ATPase activity was with the In the reverse gyrase from M. kandleri, the conserved amino acid motifs shared with other DNA-dependent ATPases are in RgyB E.R. Kozyavkin S.A. Slesarev A.I. Gellert M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 106-110Google Scholar). RgyB a DNA-dependent ATPase activity but only at temperatures hydrolysis of ATP was not until °C, with a large in activity from to 80 °C RgyB with DNA at 80 °C with ATP at temperatures not ATPase activity not RgyA by not in the presence of DNA not supercoiling activity of reconstituted RgyA and were performed at 80 °C for 15 min in the presence of EDTA, or with ATP as The figure is a of an gel with the to Image supercoiling activity was reconstituted from the recombinant RgyA and RgyB protomers by equal volumes of the subunits in storage buffer of for RgyA and for for 5 min at 80 °C, and as described 12 and of the reaction products on agarose gels in the presence of chloroquine demonstrated that either relaxed or negatively supercoiled DNA positively supercoiled 12 In the absence of but with was to and the DNA but was not to the reaction In the absence of of the supercoiled was purified from M. kandleri, the reverse gyrase heterodimer was with the two subunits through separate steps (17Kozyavkin S.A. Krah R. Gellert M. Stetter K.O. Lake J.A. Slesarev A.I. J. Biol. Chem. 1994; 269: 11081-11089Google Scholar). SDS-PAGE was the only found to separate the two subunits. To a stable heterodimer be reconstituted from the recombinant a of RgyA and RgyB was dialyzed against storage buffer at 4 °C in with separate samples of RgyA and The samples were and of each sample was heated to 80 °C for 5 min. Samples were then analyzed using gel filtration chromatography at room temperature. Both heated and samples of RgyB eluted at a with a molecular of that this subunit is monomeric at a concentration of 5 In RgyA eluted in the volume of the column After to 80 °C, approximately of the eluted at a with a The elution of the mixed sample a of the RgyA and RgyB In the elution of the heated sample that the peak corresponding to the RgyA aggregate in and the peak corresponding to RgyB shifted from to kDa SDS-PAGE analysis of fractions demonstrated that both RgyA and RgyB were in the peak in approximately a 1:1 with RgyA in the volume isolated from this peak had a activity of × which is to the activity of the enzyme from M. of mixed samples of RgyA and of a stable Samples were to overnight at 4 °C analysis using sizing fractions were collected during column elution with 10 μl of each onto the sample was directly onto the sizing sample was heated for 5 min at 80 °C onto the molecular Image of a of RgyA and RgyB at low temperature not a stable heterodimer the subunits separately at 80 °C for 5 min followed by cooling and at room temperature However, (80 °C for 5 the two subunits to the of a stable The of an active heterodimer of the two subunits at high temperature may be to the of RgyA from a that from its growth in E. coli. proteins from extreme that are in E. to by and J. F.T. 1995; demonstrated that approximately 50% of the enzyme from the is in the form of overexpressed and purified from E. coli. the these to the active To reverse gyrase from M. kandleri is the first heterodimeric enzyme from a to be (1Adams M.W.W. Perler F.B. Kelly R.M. Bio/Technology. 1995; 13: 662-668Google Scholar). that an active reverse gyrase can be reconstituted from a recombinant both analysis and can be used to the reaction of this We of laboratory for and during the of this We B. and for reading and on the