Functional and Biochemical Characterization of a Recombinant 3-Deoxy-d -manno-octulosonic Acid 8-Phosphate Synthase from the Hyperthermophilic Bacterium Aquifex aeolicus

Biochemical and Biophysical Research Communications 263, 346 –351 (1999) Article ID bbrc.1999.1361, available online at http://www.idealibrary.com on

Functional and Biochemical Characterization of a Recombinant 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase from the Hyperthermophilic Bacterium Aquifex aeolicus Henry S. Duewel, Galina Ya. Sheflyan, and Ronald W. Woodard 1 Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1065

Received July 20, 1999

The kdsA gene from the hyperthermophilic bacterium Aquifex aeolicus was cloned into a vector for expression in Escherichia coli and the kdsA gene product, 3-deoxyD-manno-octulosonic acid 8-phosphate synthase (KdsA), was overexpressed under optimized growth conditions. The thermophilic KdsA was purified using an efficient purification procedure including a heat-treatment step. Purified KdsA was shown to catalyze the formation of 3-deoxy-D-manno-octulosonic acid 8-phosphate from phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A 5-P) as determined from 1H NMR analysis of the product. Analytical gel filtration analysis indicated the native enzyme to be oligomeric. KdsA was extremely thermostable, exhibiting maximal activity at 95°C and with half-lives of 1.5 h (90°C), 8.1 h (80°C), and 30.3 h (70°C). KdsA appeared to follow Michaelis–Menton kiA5-P PEP netics with K m 5 8 2 74 mM, K m 5 43–28 mM, and k cat 5 21 0.4 –2.0 s between 60 and 90°C. © 1999 Academic Press

Lipopolysaccharide (LPS) is a fundamental constituent of the gram-negative bacterial cell envelope consisting of several distinct regions (1, 2). The inner core region of the LPS contains 2–3 residues of the unique octulose, 3-deoxy-D-manno-octulosonic acid (Kdo). Kdo serves to join Lipid A, the membrane embedded component of the LPS, to the remaining outer core and O-polysaccharide elements of the LPS. The minimal LPS structure required for the growth of most bacteria consists of lipid A derivatized with two molecules of Kdo (1). Inhibition of the synthesis of LPS with subsequent arrest in cell growth has been attributed to specific mutations in Kdo biosynthesis (3– 6). For these reasons as well as the requirement of LPS for virulence, the inhibition of Kdo metabolism and LPS bio1 To whom correspondence should be addressed at College of Pharmacy, 428 Church Street, Ann Arbor, MI 48109-1065. Fax: (734) 763-5633. E-mail: [email protected].

0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

synthesis are considered viable strategies in the development of novel antibiotics (7–9). The biosynthesis of LPS is unique to Gram-negative bacteria and although Kdo has been generally considered exclusive to the LPS, there is evidence suggesting the occurrence of Kdo in plants (10, 11). The first committed product directed towards the biosynthesis of Kdo for incorporation into the LPS is the phosphorylated precursor, Kdo 8-phosphate (Kdo 8-P). This monosaccharide is the product of the enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (KdsA; EC 4.1.2.16) which catalyzes the irreversible condensation of D-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to yield inorganic phosphate and Kdo 8-P (12, 13). Subsequent enzymes of the Kdo pathway include Kdo 8-P phosphatase and CTP:CMP-Kdo cytidylyltransferase, which produce Kdo and CMP-Kdo, respectively, the latter being the activated precursor for the enzyme Kdo-transferase which adds two Kdo residues to lipid A (2, 14 –16). Of the enzymes involved with Kdo metabolism, KdsA has received considerable attention and much of the current information accumulated for KdsA has come from studies on the enzyme from Escherichia coli (13, 17–23). Apart from preliminary accounts on the enzymes from Neisseria gonorrhoeae (24), Chlamydia psittaci (25), Pasteurella trehalosi (26) and the demonstration of KdsA activity in some plant species (10), to our knowledge, no detailed reports on KdsA from other sources have appeared. Recently, the complete nucleotide sequence of the hyperthermophilic bacterium Aquifex aeolicus, a bacterium with growth-temperature maxima near 95°C and representing one of the earliest diverging eubacteria, was determined (27). A comprehensive analysis of the genome, based on sequence homologies with other bacterial proteins of known function, has resulted in the assignment of the majority of the open reading frames contained in the genome (27). From

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this analysis, a gene showing strong similarity to the E. coli kdsA gene (28), which encodes for KdsA, was identified in the A. aeolicus genome. Our interest in KdsA from diverse organisms prompted an investigation to substantiate the predicted function ascribed to the A. aeolicus kdsA gene product. In the current study, we describe the overexpression, purification and the enzymatic properties of a newly isolated KdsA from A. aeolicus. MATERIALS AND METHODS Materials. Phosphoenolpyruvate mono(cyclohexylammonium) salt, D-arabinose 5-phosphate disodium salt and thiobarbituric acid were used as provided by Sigma Chemical Company. All other chemicals were used as supplied by Fischer. A. aeolicus genomic DNA was generously provided by Robert Huber (Lehstuhl fu¨r Mikrobiologie, Universita¨t Regensburg, Regensburg, Germany). Plasmid pT7-7 (29) was part of a laboratory stock originally supplied by Professor Stanley Tabor. Restriction endonucleases, T4 DNA ligase and reagents for performing PCR were purchased from New England Biolabs, Inc. or Boehringer Mannheim. Custom oligonucleotide primer synthesis and DNA sequencing were services provided by the University of Michigan Biomedical Research Resources Core Facility. Plasmid DNA was prepared for sequencing using the PERFECTprep Plasmid DNA Kit (5Prime 3 3Prime, Inc.). All nucleic acid manipulations were performed according to standard procedures (30) or as suggested by the manufacturer. Construction of plasmid pAakdsA. The nucleotide sequence of the open reading frame annotated as the kdsA gene was retrieved from GenBank with accession number AE000673 (g2982835). Primers P1 (59-GATTCTAGAATTCATATGGAAAAGTTTTTAGTGATAGC-39) and P2 (59-GATTCTGAATTCGGATCCAAGCTCATTTAACGGGAATTGTTTCG-39) were constructed to correspond to the 59 and 39 ends of the kdsA gene, respectively. Primer sequences complimentary to the kdsA gene are underlined. Using these primers, standard PCR methodologies were employed to amplify the kdsA gene from A. aeolicus DNA. The amplified product (841 bp) was purified from the reaction using the Wizard PCR Purification Kit (Promega) and then treated with NdeI and BamHI. The restricted fragment was isolated from 1% agarose utilizing the QIAquick Gel Extraction Kit (Qiagen) and then ligated into pT7-7 previously restricted with the same enzymes. The ligation reaction was used directly to transform competent E. coli XL 1-Blue (Stratagene). Plasmid DNAs isolated from several transformants and identified by restriction analysis to contain the PCR product were subjected to DNA sequencing to confirm the correct sequence of the kdsA gene. One plasmid with the correct sequence was named pAakdsA and was used to transform competent E. coli BL21(DE3) cells (Novagen). Overexpression and purification of KdsA. The E. coli BL21(DE3) cells harboring pAakdsA were grown in 2 3 YT medium containing ampicillin (100 mg/l) at 37°C with shaking. When the culture had reached an absorbance of 1.5 at 600 nm, solid IPTG was added to a final concentration of 0.3 mM. The culture was grown as above for an additional 8 h, after which the cells were collected by centrifugation (13000 3 g, 15 min, 4°C) and were suspended in 20 mM Tris (pH 7.5). The cell suspension was subjected to sonication on ice and then clarified by centrifugation (35000 3 g, 20 min, 4°C) to produce the cell extract. Solid sodium chloride was added to the cell extract to a final concentration of 0.1 M and the solution was heated in a boiling water bath for 1.5 min and then at 80°C for 10 min with continuous swirling. The suspension was allowed to cool to room temperature and then placed on ice for 15 min. Precipitated protein was removed

by centrifugation (30000 3 g, 20 min, 4°C) and the supernatant was dialyzed against 20 mM Tris (pH 7.5). Ten to fifteen-milligram quantities of the dialyzed heat-treated protein were applied to a Mono Q column (HR 10/10; Pharmacia) previously equilibrated with 20 mM Tris (pH 7.5). The column was developed at a flow rate of 2 ml/min using a linear gradient to 0.3 M potassium chloride in the same buffer (over 40 min). KdsA activity resolved into a single peak (approximately 30 to 35 min into the gradient) and fractions containing KdsA were pooled, concentrated by ultrafiltration (Centriprep-10; Amicon) and then dialyzed against 20 mM Tris (pH 7.5). The purified enzyme was aliquoted and frozen in liquid nitrogen and stored at 280°C. KdsA activity assays. Temperature control for enzymatic assays was achieved with use of a programmable thermal controller (MJ Research, Inc.). All assays were performed in triplicate. A unit of enzyme activity is defined as the production of 1 mmol of Kdo 8-P per minute. Standard assay. Enzyme activity was measured in a final volume of 150 ml containing PEP (3 mM), A 5-P (3 mM), Tris-acetate buffer (100 mM, pH 7.5) and using thin-walled PCR tubes (United Laboratory Plastics) as the reaction vessel. The assay solution was preincubated at a desired temperature (2 min) and the reaction was initiated with the addition of enzyme (5 ml) and incubated at the desired temperature. At specified times, the reactions were stopped with the addition of ice-cold TCA (to a final concentration of 5%) and then centrifuged to remove protein. The amount of Kdo 8-P produced was determined by subjecting aliquots of the quenched reactions to the periodate-thiobarbituric acid assay as specified by Ray (31). Temperature optima of purified KdsA. The temperature optimum of KdsA was determined by measuring the activity between 30 and 100°C using the standard assay and 0.65 mM purified enzyme. Reactions were incubated at the respective temperatures for 5 min. Thermostability of purified KdsA. Purified enzyme (10 mM) in 100 mM Tris-acetate (pH 7.5) was constantly maintained at 70, 80 or 90°C in thin-walled PCR tubes. At various times, aliquots of enzyme (10 ml) were removed and subjected to the standard assay. Reactions were incubated for 5 min at the corresponding initial incubation temperature of the enzyme. Kinetic constants. Reactions were performed separately at 60, 70, 80 and 90°C under standard assay conditions with the following exceptions. Initial velocities were determined using at least eight concentrations of A 5-P in the presence of 1 mM PEP and 0.13 mM purified enzyme. Similarly, reactions were performed using at least eight concentrations of PEP in the presence of 3 mM A 5-P. In all cases, reactions were stopped after a 2-min incubation. Samples using the highest substrate concentrations but without enzyme served as the blanks. Values for K m and k cat were obtained by fitting plots of velocity against substrate concentration according to the Michaelis-Menton equation with the assumption that the initial rate was linear over the 2-min period. Molecular weight determinations. The subunit molecular weight of KdsA was determined by electrospray ionization mass spectrometry at the University of Michigan Biomedical Research Resources Core Facility on a Micromass Platform instrument equipped with an electrospray ionization source. The protein sample was exchanged into water by ultrafiltration prior to analysis. The native molecular weight of KdsA was determined by analytical gel filtration chromatography using a Superose 12 column (HR 10/30; Pharmacia). Chromatography was performed at ambient temperature on a Pharmacia FPLC system monitoring the eluent at 280 nm. The running buffer was 0.15 M sodium chloride, 50 mM Tris (pH 7.5) and a flow rate of 0.5 ml/min was used. A standard curve was generated using proteins from a molecular weight marker kit (MWGF-200, Sigma) as outlined by the manufacturer. Blue dextran 2000 served to determine the column void volume, V o (6.13 6 0.03 ml), for the particular column used. Purified E. coli KdsA, isolated as previ-

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Purification of KdsA from A. aeolicus a Step

Vol (ml)

Protein (mg)

Activity (U) b

Specific activity (U mg 21) b

Recovery (%)

Purification fold

Cell extract Heat treatment Mono Q

43.0 45.0 19.3

205 45.0 22.4

162.0 128.0 75.6

0.79 2.84 3.38

100 79 47

1 3.6 4.3

a b

Purification was achieved from a 1-liter culture of E. coli BL21(DE3) harboring pAakdsA. Determined at 80°C.

ously described (18), was also subjected to analysis under identical conditions. The molecular weight of the E. coli enzyme was calculated to be 30833 based on the primary sequence (28). Standards and samples were used at concentrations of 0.5–1.0 mg/ml and were loaded onto the column using a 50 ml sample loop. The elution volume, V e, was determined in triplicate for all samples and standards. Semi-preparative synthesis of Kdo 8-P. Purified enzyme (35 mM) was incubated in the presence of PEP (14 mM), A 5-P (28 mM) and Tris-acetate buffer (100 mM, pH 7.5) in a final volume of 2.22 ml. The reaction was incubated for 15 min at 80°C and then quenched with the addition of 40% TCA (0.2 ml) and precipitated protein was removed by centrifugation. The 3-deoxy-a-keto sugar acid component of the reaction mixture was isolated by anion exchange chromatography over an Econo-Pac High Q cartridge (BioRad) and the 500 MHz 1H NMR spectrum of the isolate was acquired as previously described (32). Miscellaneous methods. Optical spectroscopy was performed on a Cary 3 Bio UV-Visible Spectrophotometer (Varian Associates). SDSPAGE was performed with a Mini-PROTEAN II electrophoresis unit (Bio-Rad). Protein concentrations were estimated using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad) with bovine serum albumin (Sigma) serving as the calibration standard.

RESULTS AND DISCUSSION This report describes the purification and initial characterization of KdsA from A. aeolicus overexpressed in E. coli and to our knowledge, represents the first account on a recombinant protein from this extreme hyperthermophile. PCR was utilized to amplify the kdsA gene from A. aeolicus genomic DNA and cloning of the kdsA gene into plasmid pT7-7 permitted expression of the A. aeolicus KdsA in E. coli BL21(DE3) harboring plasmid pAakdsA. We have previously used the pT7-7 vector and E. coli BL21(DE3) expression system to achieve excellent overproduction (.100 mg per liter of culture) of E. coli KdsA (18). However, initial studies had indicated significantly reduced expression levels of A. aeolicus KdsA, most likely resulting from non-optimal codon usage for the translation of the thermophilic kdsA gene in E. coli. Induction of KdsA expression from E. coli BL21(DE3) harboring pAakdsA was therefore explored under several conditions by varying both the cell density of the culture at the time of induction with IPTG and the post-induction growth. SDS-PAGE analysis (not shown) of whole cells taken from cultures treated

under the various conditions indicated that maximal expression of KdsA was achieved from cells induced at an absorbance of 1.5 at 600 nm and then grown for an additional 8 –12 h post-induction period. Under these conditions, KdsA constitutes approximately 30% of the total cellular protein (Fig. 1, lane 3). The conditions developed for cell growth, though optimized to overexpress the foreign kdsA gene in E. coli, produced a high level of contaminating host cell proteins. It was determined that heat-treatment of the cell extract at 60, 70 or 80°C for 10 min resulted in substantial precipitation of the host proteins while KdsA remained soluble. The greatest quantity of host proteins was eliminated at 80°C and treatment of the cell extract at this temperature in excess of 10 min did not improve the degree of purification. The addition of 0.1 M sodium chloride to the cell extract prior to heat-treatment significantly assisted in the removal of the impurities by centrifugation and improved the quality of the resulting fraction for subsequent chromatography. A summary of the purification of KdsA is presented in Table 1 and Fig. 1. Using the protocol developed, KdsA was isolated in greater than 98% purity (Fig. 1, lane 7) and consistently yielded 20 –25 mg of purified protein per liter of cell culture. Treatment of the cell extract at 80°C for 10 min proved very effective as an initial purification step. The majority of contaminating

FIG. 1. SDS-PAGE analysis of the stages of purification of KdsA. Analysis was performed under reducing conditions on a 12% polyacrylamide gel and visualized with Coomassie R-250 stain. Lane 1, molecular weight standards; lane 2, whole cells without IPTG induction; lane 3, whole cells with IPTG induction; lane 4, cell free extract (25 mg); lane 5, heat-treatment pellet fraction; lane 6, heat-treatment soluble fraction (10 mg); lane 7, Mono-Q fraction after dialysis and concentration (10 mg). KdsA corresponds to the band migrating at an apparent molecular weight of 33 3 10 3.

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FIG. 2. Determination of the quaternary structure of KdsA at 25°C by analytical gel filtration analysis on a Superose 12 column. The elution parameter, V e/V o, was determined for each protein and is plotted against the logarithm of the molecular weight of the standard proteins (E), or of the molecular weight calculated from the primary sequence and assuming a dimeric (59468 Da), trimeric (89202 Da), or tetrameric (118936 Da) structure for A. aeolicus KdsA (F). Similarly, V e/V o determined for E. coli KdsA (h) was plotted against the logarithm of the dimeric (61666), trimeric (92499 Da) or tetrameric (123332) molecular weight for E. coli KdsA. Standards used were as follows: cytochrome c (12.4 3 10 3); carbonic anhydrase (29.0 3 10 3); albumin (66.0 3 10 3); alcohol dehydrogenase (141 3 10 3); and b-amylase (200 3 10 3).

protein was removed by application of the heattreatment step (Fig. 1, lanes 5 and 6) with minimal loss of KdsA activity (Table 1). The molecular weight of the purified recombinant enzyme was 29736 6 1 as determined by electrospray ionization mass spectrometry. This value is in excellent agreement with a calculated molecular weight of 29734 based on the open reading frame coding for the KdsA protein (267 amino acids). The quaternary structure of KdsA was estimated by analytical gel filtration chromatography. Figure 2 summarizes the results obtained on a calibrated Superose 12 column. A plot of V e/V o versus log (M r) for the standard proteins yielded a straight line (R 5 0.993). When V e/V o for KdsA (1.845 6 0.008) was plotted versus the log (M r) calculated for several oligomeric states, closest fits to the curve were obtained for either a trimeric (calcd. M r 5 89.2 3 10 3 ) or tetrameric (calcd. M r 5 119 3 10 3 ) structure with the trimer giving the overall best fit. The apparent molecular weight for KdsA interpolated from the curve was 97.6 3 10 3, 3.3 times the molecular weight calculated for the primary structure. These results suggest that the native enzyme could either be an elongated trimer or a compact tetramer in solution, or could indicate a rapid trimer-tetramer equilibrium under the conditions examined. For comparative purposes, E. coli KdsA was also subjected to similar analysis. Within error, chromatography of the E. coli enzyme produced a V e/V o value (1.837 6 0.010) equivalent to that of the thermophilic

KdsA (Fig. 2). The identical migration of the two proteins under the conditions of gel filtration strongly suggests analogous native molecular weights and oligomeric structures considering that the calculated primary molecular weight of KdsA from E. coli (M r 5 30833) is only slightly larger than that of the A. aeolicus enzyme. Previous estimates of the native molecular weight of the E. coli enzyme by Ray (13) had indicated a value of 90.0 3 10 3, leading to the proposal of a trimeric structure for the E. coli KdsA. Further studies including chemical crosslinking experiments will be completed to provide additional insight onto the oligomeric structure of the two enzymes. The most important objective of this study was to unambiguously establish the catalytic function of KdsA from A. aeolicus through direct characterization of the product formed upon incubation of purified enzyme with PEP and A 5-P. To this end, a semipreparative scale reaction was performed and the 3-deoxy-monosaccharide product, as determined by the periodate-TBA assay, was purified from the reaction components by anion exchange chromatography and characterized by NMR. Although it has been generally accepted that the presence of a 3-deoxy-a-keto sugar acid can be detected by the periodate-TBA assay, this method does not provide any information with respect to the stereochemistry of the reacting monosaccharide. In the condensation reaction between PEP and A 5-P, C3 of PEP could attack either the re face of the carbonyl of A 5-P to give 3-deoxy-D-manno-octulosonic acid 8-P (i.e. Kdo 8-P), or attack can occur on the si face of the carbonyl which would produce 3-deoxy-D-glucooctulosonic acid 8-P (the difference lies in the stereochemistry at C4 of the product). We consider the characterization of the condensation product by NMR as the only reliable approach to establish the true identity of the product. The 500 MHz 1H NMR spectrum of the product isolated (spectrum not shown) was in complete agreement with that reported previously for 3-deoxyD-manno-octulosonic acid 8-phosphate (17, 32). This result establishes that the recombinant protein under investigation does in fact catalyze the formation of Kdo 8-P via attack of PEP on the re face of A 5-P. In addition, the function assigned to the A. aeolicus kdsA gene product from the original genomic analysis (27), has been shown to be correct. The effect of temperature on the specific activity of A. aeolicus KdsA is illustrated in Fig. 3. Enzymatic activity increased with increasing temperature with maximal activity observed at 95°C. Between the temperatures of 60 and 95°C, the dependence of specific activity on temperature was essentially linear. Pretreatment of the enzyme at 80°C for 5 min prior to the determination of activity at lower temperatures did not have any effect on the activity measured. The specific activity of the enzyme at 95°C (5.4 6 0.1 U mg 21) was 54-fold higher than that of the activity measured at 30°C

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FIG. 3. Determination of optimum temperature of KdsA activity. Samples of KdsA were incubated for 5 min at the indicated temperatures in the presence of PEP and A 5-P in 100 mM Tris–acetate (pH 7.5) according to the standard assay. The concentration of KdsA during the assay was 0.65 mM.

FIG. 4. Thermal stability of KdsA activity. Enzyme (10 mM) was incubated in 100 mM Tris–acetate buffer (pH 7.5) at 70 (F), 80 (■), or 90°C (}). At various intervals, aliquots of the samples were removed and assayed for residual activity at the respective incubation temperature.

(0.1 6 0.02 U mg 21). This maximal activity compares well to the E. coli enzyme for which values of 3.7 to 9 U mg 21 have been determined under similar conditions but at lower temperatures (13, 21). The enzymatic constants of KdsA were determined for A 5-P and PEP under saturation conditions (Table 2). KdsA appeared to follow Michaelis-Menton kinetics. As in the case with the specific activity, k cat increased linearly between the temperatures examined at a rate of approximately 0.05 s 21 per degree. The apparent binding of A 5-P decreased marginally with increasing temperature while PEP binding was not greatly affected as reflected in the K m values for A 5-P and PEP, respectively. The apparent K m values for A 5-P and PEP determined for the A. aeolicus KdsA again compare favorably with those reported for the E. coli enzyme determined at 37°C (K mPEP ' 6 mM; K mA5-P ' 20 mM) (13, 20, 23). The energy of activation as determined according to the Arrhenius equation from a plot of log(k cat) vs 1/T (33) was calculated to be 13.4 6 1.3 kcal mol 21. This value is in excellent agreement with an energy of activation of 15 kcal z mol 21 reported for KdsA from E. coli and, as previously noted, may be related to the energy of hydrolysis of PEP (13).

With the knowledge that the properties of the recombinant KdsA isolated in the current study resemble those of the E. coli enzyme, we are confident that overexpression of the A. aeolicus KdsA in E. coli produced a functionally competent and correctly folded enzyme. More conclusive evidence that the recombinant KdsA was produced and isolated in its natural state is evident from the exceptional thermostability demonstrated by the enzyme. Figure 4 illustrates the effect of temperature on enzyme activity as a function of time. KdsA activity appeared to decrease exponentially with time at each respective temperature examined with calculated half-lives of 1.5 h at 90°C, 8.1 h at 80°C and 30.3 h at 70°C. Analytical gel filtration analysis of purified enzyme maintained at 90°C over time indicated that thermal inactivation of KdsA results from denaturation of the intact oligomer and not from dissociation of the oligomer to transient dimeric or monomeric species (not shown). The level of stability demonstrated by the A. aeolicus KdsA vastly exceeds those reported for other KdsA homologs (10, 13) and falls into a range of the extraordinary thermostability exhibited by other proteins isolated from hyperthermophilic organisms (34, 35). We have taken advantage of the enzyme’s tolerance to high temperatures during purification and have achieved a high level of purity for the enzyme through simple heat-treatment of the cellular extract. The general application of a heattreatment step may prove useful towards the purification of other recombinant proteins from this organism. Currently, direct evidence supporting the existence of an LPS in A. aeolicus is lacking. We have unequivocally demonstrated by means of 1H NMR analysis that the recombinant A. aeolicus KdsA catalyzes the formation of Kdo 8-P upon incubation with PEP and A

TABLE 2

Kinetic Constants of KdsA at Various Temperatures Temp (°C)

k cat (s 21)

60 70 80 90

0.38 6 0.08 0.87 6 0.01 1.47 6 0.05 2.00 6 0.11

K mPEP (mM)

K mA5-P (mM)

43 6 6 43 6 5 38 6 5 28 6 4

861 15 6 1 27 6 4 74 6 8

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5-P. This result indicates that A. aeolicus is capable of producing Kdo 8-P, the precursor that in other Gramnegative bacteria, serves as the first committed product in LPS biosynthesis. Furthermore, putative genes involved in different stages of LPS biosynthesis have also been identified in the A. aeolicus genome, including lpxB (lipid A disaccharide synthetase), kdtA (Kdo transferase), kpsU (CTP:CMP-Kdo cytidylyltransferase) and kdtB (lipopolysaccharide core biosynthesis protein) (27). Although these observations do not provide direct evidence confirming the incorporation of Kdo as an integral component of the A. aeolicus LPS or indeed the presence of LPS in this organism, they are strongly supportive thereof. In summary, A. aeolicus KdsA represents only the second member of the KdsA enzyme family and the first recombinant protein from A. aeolicus that has been purified and characterized in any detail. The results presented here have shown that A. aeolicus KdsA shares characteristics in common with its mesophilic counterpart from E. coli. Further studies will attempt to take advantage of the distinctive thermostable properties of A. aeolicus KdsA and may contribute to understanding the key mechanistic and biochemical features of this emerging enzyme family. ACKNOWLEDGMENTS The authors are grateful to Robert Huber for the generous gift of A. aeolicus DNA. The excellent technical assistance of Ms. Dana Randall during the early stages of purification is appreciatively noted. This work was supported by NIH Grant GM53069 with partial support from an NSERC (Canada) postdoctoral fellowship (H.S.D.).

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