Reverse Gyrase from Methanopyrus kandleri
RECONSTITUTION OF AN ACTIVE EXTREMOZYME FROM ITS TWO RECOMBINANT SUBUNITS*

(Received for publication, November 8, 1996, and in revised form, March 9, 1997)

Regis Krah , Mary H. O'Dea and Martin Gellert Dagger

From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0540

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

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 (1). However, many archaea fail to grow in the laboratory (2), and the majority that can be grown do so inefficiently (3). 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 (1, 4). 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 (1).

Reverse DNA gyrases, a novel class of type I 5'-topoisomerases, were discovered in hyperthermophiles (5-11). 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 (12, 13). Reverse gyrase is thus considered a molecular marker for hyperthermophilic prokaryotes (14). 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 (12, 13). Sequence analysis of reverse gyrase from Sulfolobus acidocaldarius revealed that the type I 5'-topoisomerase domain was fused to a domain reminiscent of a nucleic acid helicase (10). Helicases are nucleic acid-dependent ATPases as is reverse gyrase (15). 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 (16-18). 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 (17). 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 from E. coli and the reconstitution of active, stable reverse gyrase from the purified proteins.


EXPERIMENTAL PROCEDURES

Materials

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 (19). Isopropyl-1-thio-beta -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)1 was from Calbiochem. Phosphocellulose P-11 was from Whatman. Both L-broth and SuperbrothMDRV were purchased from Advanced Biotechnologies Inc.

Methods

Column Chromatography, Protein Gel Electrophoresis, and Determination of Protein Purity

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.

Assay of ATPase Activity

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 [gamma -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 0.75 M KH2PO4. Radioactivity was quantitated with a PhosphorImager (Molecular Dynamics, Inc.).

Reconstitution of Reverse Gyrase and Assay for Topoisomerase Activity

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.

Construction of Expression Vectors

RgyA and RgyB were produced separately in E. coli using the pET expression system of Studier et al. (20). The rgyB coding sequence was amplified by polymerase chain reaction from a recombinant lambda  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 lambda  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.

Protein Purification

Four liters of Superbroth were inoculated with BL21::lambda DE3 pLysE carrying the RgyA or RgyB expression plasmid. Cells were grown at 37 °C to an A600 of 0.8 with aeration, then isopropyl-1-thio-beta -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 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.

Purification of RgyA

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 mM Tris, 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% glycerol), and used in subsequent experiments. The pooled fractions of peak 2 yielded 2.7 mg of protein that was >90% pure (Table I).


Fig. 1. Purification of RgyA. Details of the purification are under "Experimental Procedures." Lane 1, cleared lysate; lane 2, 1 M NaCl polyethyleneimine extract (Poly-P); lane 3, (NH4)2SO4 fraction; lane 4, low salt RgyA precipitate resolubilized in buffer with 1 M NaCl; lanes 5-15, HTH column fractions. Samples were diluted 1:3 with water before the addition of an equal volume of 2 × Tris/glycine SDS sample buffer. 10 µl of this sample was loaded into each lane.
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Table I. Purification of reverse gyrase subunits


Step Volume Total Protein

ml mg
RgyA
  Cell paste 16,000
  Cleared lysate 120 1,007
  Polyethyleneimine 48 1128
  (NH4)2SO4 28 293
  Dialysis 15 67
  HTH pool 6 2.8
RgyB
  Cell paste 16,000
  Cleared lysate 90 1,476
  Polyethyleneimine 50 991
  (NH4)2SO4 22 635
  Ni2+ column 8.0 81
  Dialysis 9.5 32
  Heparin pool 5 8
  HTH pool 2 2
  S200 pool 4.5 0.62

Purification of RgyB

The ammonium sulfate-precipitated RgyB was resuspended in 20 ml of buffer C (50 mM Tris, pH 8, 1 M NaCl, 2 mM beta -mercaptoethanol) containing 10 mM imidazole and 0.1 mM EDTA, pumped through a 5-ml phosphocellulose column, and loaded directly onto a 3-ml Ni2+-Sepharose column. The phosphocellulose column was removed, and the Ni2+ column was washed with 75 ml of buffer C containing 0.05 M imidazole followed by a gradient from 0.05 to 2 M imidazole in buffer C (30 ml total). Fractions containing RgyB were pooled and dialyzed overnight against 2 liters of buffer D (50 mM Tris, pH 8, 100 mM NaCl, 0.1 mM EDTA, and 2 mM DTT). Some precipitate was observed that was removed by centrifugation. The sample was loaded onto a 5-ml HiTrap heparin column at 0.1 ml/min and washed with 25 ml of buffer D, and the protein was eluted with a linear gradient of 0.1-1 M NaCl in buffer D (50 ml total, Fig. 2). Fractions in which the major protein observed was full-length RgyB were pooled, adjusted to 10 mM potassium phosphate, and loaded directly onto a 2-ml HTH column, and the sample was washed with 10 ml of Buffer E (50 mM Tris, pH 8, 200 mM NaCl, 10% glycerol) plus 10 mM potassium phosphate. The protein was eluted with a linear gradient (20 ml total) from 10 to 600 mM potassium phosphate, pH 7.4, in buffer E. Fractions in which the major protein component was full-length RgyB were pooled. The sample was split into three separate aliquots, and each aliquot was separated on a Superdex 200HR 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 0.61 mg of protein (Fig. 2, Table I).


Fig. 2. Purification of RgyB. Details of the purification are under "Experimental Procedures." Solid bars above gel lanes indicate which fractions were pooled before proceeding to the next step. By careful selection of column fractions during purification, it was possible to obtain a preparation containing almost exclusively full-length RgyB. A, heparin column. The load for this column is the pooled sample from the Ni2+-Sepharose column. Fractions taken from this column eluted near a salt concentration of 350 mM NaCl. MWM, molecular mass standards. B, HTH column. The pooled fractions from the column eluted near 300 mM potassium phosphate. C, gel filtration S200 sizing chromatography.
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RESULTS AND DISCUSSION

Production of Recombinant RgyA and RgyB in E. coli

RgyA protein was insoluble when overexpressed in E. coli at 37 °C, but solubility was improved by expressing the protein at 20 °C. After induction of the cells and lysis as described under "Experimental Procedures," approximately 50% of the overexpressed protein remained in the soluble fraction of the cleared lysate. The same proportion of RgyA stayed in the soluble fraction even when the salt concentration of the lysis buffer was lowered to 100 mM NaCl. The production of soluble RgyA may rely on the cold shock response of E. coli. Alternatively, translation at a lower temperature may proceed slowly enough to allow regions of secondary structure sufficient time to fold. Improved solubility of other recombinant proteins from hyperthermophiles by growth at lower temperatures has been observed (1). The solubility of recombinant RgyB was also improved by expressing the protein at 20 °C (data not shown).

RgyB undergoes considerable proteolytic cleavage and premature translation termination during expression in E. coli, creating a very heterogeneous sample (Fig. 2A, lane 2). However, by being selective in which fractions were pooled during purification, it was possible to isolate a sample that was almost exclusively full-length RgyB.

Using a single-column purification protocol for RgyA and a four-column purification protocol for RgyB, each subunit was purified to >90% homogeneity as determined by scanning analysis of stained SDS-PAGE gels of each sample.

Analysis of RgyA

Although RgyA eluted as two separate peaks from an HTH column (Fig. 1), these two fractions had biochemical properties that were indistinguishable by several tests. In a supercoiling assay of 1 µl of each column fraction of RgyA from the HTH column together with 0. 25 µg of purified RgyB revealed two peaks of enzymatic activity corresponding with the two peaks of RgyA protein that eluted from the column. RgyA samples from peak 1 and peak 2 both elute in the excluded volume of an S200 sizing column, indicating a tendency to form high molecular mass aggregates (see below). Finally, protein from either peak of RgyA can form a stable heterodimer with RgyB at high temperature (see below). We speculate that these two peaks may represent two different folding intermediates of RgyA that have different affinity for the HTH packing material.

In the early steps of the purification procedure, RgyA had a strong tendency to precipitate out of solution at lower salt concentrations. We used this property to our advantage in the purification by collecting the precipitate that formed during dialysis from 1 M NaCl to 300 mM NaCl (Fig. 1). This precipitate readily dissolved in buffers containing 1 M NaCl and did not behave like denatured protein. After passage of the sample through a phosphocellulose column, RgyA remained in solution on dialysis into buffers containing as little as 100 mM NaCl. We believe this precipitation of early fractions is due to residual polyethyleneimine in the sample that is later removed by passage through the phosphocellulose column (19).

RgyB Is a DNA-dependent ATPase

Because all previously isolated reverse gyrases were single-chain enzymes (14, 15), it had not been possible to test whether the DNA-dependent ATPase activity was associated with the helicase-like domain. In the reverse gyrase from M. kandleri, the conserved amino acid motifs shared with other DNA-dependent ATPases are in RgyB (18). Recombinant RgyB displayed a DNA-dependent ATPase activity but only at elevated temperatures (Fig. 3). Efficient hydrolysis of ATP was not observed until 60 °C, with a large increase in activity from 60 to 80 °C (Fig. 4B). Preheating RgyB with DNA at 80 °C before incubation with ATP at lower temperatures did not enhance ATPase activity (data not shown). RgyA by itself did not hydrolyze ATP, even in the presence of DNA (data not shown).


Fig. 3. ATPase activity of RgyB. The standard reaction was a 20-µl volume with 0.1 µg of RgyB, 0.1 µg of plasmid DNA, and 50 µM [gamma -32P]ATP. A, hydrolysis of ATP at 80 °C as a function of time. The lowest curve (DNA) represents the spontaneous hydrolysis of ATP at this temperature. B, temperature dependence of ATP hydrolysis by RgyB with spontaneous hydrolysis of ATP subtracted. Reactions were incubated for 15 min.
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Fig. 4. Positive supercoiling activity of reconstituted RgyA and RgyB. Reactions were performed at 80 °C for 15 min in the presence of EDTA, Mg2+, or Mg2+ with ATP as indicated. The figure is a digitized photograph of an ethidium bromide-stained gel with the image inverted to enhance contrast.
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Reconstitution of Reverse Gyrase Activity

Positive supercoiling activity was reconstituted from the recombinant RgyA and RgyB protomers by mixing equal volumes of the subunits in storage buffer (at concentrations of 0.47 mg/ml for RgyA and 0.14 mg/ml for RgyB), heating for 5 min at 80 °C, and assaying as described (Fig. 4, lanes 12 and 24). Analysis of the reaction products on agarose gels in the presence of chloroquine demonstrated that either relaxed or negatively supercoiled DNA became positively supercoiled (Fig. 4, lower panel, lanes 12 and 24). In the absence of ATP, but with Mg2+ present, RgyA/B was able to bind and nick the substrate DNA but was not able to complete the ligation reaction (Fig. 4, lower panel (Relaxed Substrate), compare lanes 22-24). In the absence of Mg2+, RgyA/B nicking of the supercoiled substrate was greatly reduced.

Recombinant RgyA and RgyB Form a Heterodimer after Heating

When purified from M. kandleri, the reverse gyrase heterodimer was very stable, with the two subunits copurifying through four separate chromatographic steps (17). Denaturing SDS-PAGE was the only effective method found to separate the two subunits. To determine if a stable heterodimer could be reconstituted from the recombinant subunits, a mixture of RgyA and RgyB was dialyzed against storage buffer at 4 °C overnight, in parallel with separate samples of RgyA and RgyB. The samples were split, and half 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 unheated samples of RgyB eluted at a position consistent with a molecular mass of 150 kDa, indicating that this subunit is monomeric at a concentration of 5 µg/ml (Fig. 5). In contrast, unheated RgyA eluted in the excluded volume of the S200 column (Fig. 5; see also Fig. 6). After heating to 80 °C, approximately 20% of the material eluted at a position consistent with a monomer (40 kDa). The elution profile of the unheated mixed RgyA/B sample resembled a superposition of the unheated RgyA and RgyB profiles. In contrast, the elution profile of the heated RgyA/B sample showed that the peak corresponding to the RgyA aggregate decreased in amount, and the peak corresponding to RgyB shifted from 150 to 180 kDa (Fig. 5). SDS-PAGE analysis of fractions demonstrated that both RgyA and RgyB were present in the 180-kDa peak in approximately a 1:1 ratio, with very little RgyA in the excluded volume (Fig. 6). Enzyme isolated from this peak had a specific activity of 2.1 × 105 units/mg, which is very similar to the activity of the complete enzyme prepared from M. kandleri.


Fig. 5. Analytical gel chromatography of RgyA and RgyB. Sizing chromatography of RgyA and RgyB and the RgyA/B heterodimer using a Smart system Superdex 200 3.2/30 column.
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Fig. 6. The effect of heating mixed samples of RgyA and RgyB. Heating allows formation of a stable RgyA/B heterodimer. Samples were allowed to mix overnight at 4 °C before analysis using S200 sizing chromatography. 50-µl fractions were collected during column elution with 10 µl of each fraction loaded onto the gel. Unheated, sample was loaded directly onto the sizing column. Heated, sample was heated for 5 min at 80 °C before loading onto the column. MWM, molecular mass standards.
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Prolonged incubation of a mixture of RgyA and RgyB at low temperature did not yield a stable heterodimer (Fig. 6) nor did heating the subunits separately at 80 °C for 5 min followed by cooling and mixing at room temperature (see Fig. 7). However, heating (80 °C for 5 min) the two subunits together led to the formation of a stable heterodimer. The formation of an active heterodimer upon coincubation of the two subunits at high temperature may be due to the release of RgyA from a misfolded state that resulted from its growth in E. coli. Other proteins from extreme thermophiles that are multimers when expressed in E. coli have also required heating to become active. Work by DiRuggiero and Robb (21) demonstrated that approximately 50% of the hexameric enzyme glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus is in the form of inactive monomers when overexpressed and purified from E. coli. Heating the inactive monomers converts these to the active multimeric complex.


Fig. 7. Heating RgyA and RgyB together is required for the formation of a stable heterodimer. Solid line, RgyA and RgyB heated separately for 5 min at 80 °C then mixed at room temperature before loading onto the sizing column. Dotted line, RgyA and RgyB mixed, heated for 5 min at 80 °C, and then loaded onto the sizing column.
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To our knowledge, reverse gyrase from M. kandleri is the first heterodimeric enzyme from a hyperthermophile to be reconstituted in vitro (1). Now that an active reverse gyrase can be reconstituted from a recombinant source, both deletion analysis and site-directed mutagenesis can be used to investigate the reaction mechanism of this unusual enzyme.


FOOTNOTES

*   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.
Dagger    To whom correspondence should be addressed. Tel.: 301-496-5888; Fax: 301-496-0201.
1   The abbreviations used are: HTH, hydroxylapatite; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.

ACKNOWLEDGEMENTS

We thank members of our laboratory for helpful and insightful discussions during the course of this work. We also thank Alison B. Hickman and Joanne Hesse for reading and helpful comments on the manuscript.


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