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An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 370 (2007) 142–146 www.elsevier.com/locate/yabio
An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase Yanyan Li, Philippe Lopez, Philippe Durand, Jamal Ouazzani, Bernard Badet, Marie-Ange Badet-Denisot * Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-Sur-Yvette, France Received 12 April 2007 Available online 9 August 2007
Abstract An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-coenzyme A (CoA) to glucosamine-6-phosphate, releasing coenzyme A. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,5 0 -dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS. 2007 Elsevier Inc. All rights reserved. Keywords: Glucosamine-6-phosphate synthase; Acetyltransferase; Ellman’s reagent; Enzymatic test
Hexosamine biosynthesis is ubiquitous in all organisms and the end products of this pathway are essential sugar building blocks for biosynthesis of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals [1]. The first and rate-limiting reaction is catalyzed by glucosamine6-phosphate synthase (GlmS, EC 2.6.1.16),1 which converts D-fructose-6-phosphate (Fru-6P) into D-glucosamine-6-phosphate (GlcN6P) with hydrolysis of L-glutamine to L-glutamate. As an important enzyme controlling the amino sugar metabolism, GlmS is proposed to be a thera-
*
Corresponding author. Fax: +33 1 69077247. E-mail address: [email protected] (M.-A. Badet-Denisot). 1 Abbreviations used: Ac, acetyl; ADGP, 2-amino-2-deoxy-D-glucitol-6phosphate; ADMP, 2-amino-2-deoxy-D-mannitol-6-phosphate; DTNB, 5,5 0 -dithio-bis(2-nitrobenzoic acid); Fru-6P, D-fructose-6-phosphate; GlmS, bacterial glucosamine-6-phosphate synthase; GlcN6P, glucosamine-6-phosphate; GNA1, yeast glucosamine-6-phosphate N-acetyltransferase 1; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; TNB, 2-nitrobenzoic acid; DTT, dithiothreitol. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.07.031
peutic target in the development of antifungal [2], and more recently, antidiabetic compounds [3]. GlmS belongs to the N-terminal nucleophilic glutaminedependent amidotransferase family [4]. Structural studies have shown that GlmS from Escherichia coli possesses an intramolecular tunnel through which ammonia is transferred from the glutamine site to the Fru-6P binding site for amination [5]. Detailed investigations on the functioning of the tunnel, such as its blockage or perforation, require in particular knowledge of the amidotransferase activity (GlcN6P synthesizing activity) and thus reliable quantification of GlcN6P. Enzyme activity of GlmS was conveniently measured by quantification of glutamate using glutamate dehydrogenase as coupling enzyme. However, this method has limits in screening inhibitors of the Fru-6P binding site, since glutamate production is not always coupled to amidotransferase activity [6]. Several methods were developed for monitoring GlcN6P formation, including a colorimetric Morgan– Elson assay [7], which was adapted to microtiter plate format for inhibitor screening [8], a fluorometric assay with o-phthaldialdehyde derivatization [9], and a MALDI-TOF
Coupled assay for glucosamine-6P quantification / Y. Li et al. / Anal. Biochem. 370 (2007) 142–146
mass spectrometry-based assay [10]. They are all noncontinuous assays and require multiple derivatization steps. Recently a radiometric assay using 14C-radiolabeled Fru6P, which allows sensitive detection of GlcN6P down to 1 pmol and can be adapted to 96-well plate format, was developed [11]. However handling of radioactivity requires special caution. Here we report a simple and rapid assay for GlcN6P detection using yeast GlcN6P N-acetyltransferase 1 (GNA1) as coupling enzyme (Fig. 1). In eukaryotes, GNA1 transfers the acetyl moiety from acetyl-CoA (AcCoA) to GlcN6P, producing N-acetyl-GlcN6P and coenzyme A (CoASH) [12]. The free sulfhydryl group of CoASH can be then quantified by the widely used Ellman’s reagent, 5,5 0 -dithio-bis-(2-nitrobenzoic acid) (DTNB) in a discontinuous manner [13]. Alternatively, GlcN6P formation is followed by the disappearance of the thioester bond of Ac-CoA at 230 nm [14]. This is the first continuous method that allows monitoring GlcN6P production directly. Validation of the assay by determining the kinetic parameters of GlmS from E. coli and inhibition constants (Ki) of two inhibitors is presented in this report. Materials and methods General materials Chemicals were purchased from Sigma Chemical Co. and were of highest purity available. L-Gln was further purified by recrystallization. The 96-well PCR plates (Thermo-Fast 96, nonskirted) and adhesive PCR films were from Abgene (UK). The 96-well flat-bottomed polystyrene plates for UV measurement were from Becton–Dickinson Labware (USA). GlmS was purified and stored in buffer (50 mM Bis-Tris-propane, pH 7.2, 1 mM DTT, 1 mM EDTA, 10% glycerol) at 80 C as previously described [15]. Overexpression and purification of GNA1 Plasmid pET28-gna1 for expressing yeast GNA1 with a His6 tag was a kind gift of Dr. D. Mengin-Lecreulx (Universite´ Paris-Sud, Orsay, France) [16]. E. coli BL21(DE3)-
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pLyS cells harboring pET28-gna1 were grown at 37 C with agitation until OD600 reached 0.7–1. Protein expression was induced with 0.2 mM isopropyl-L-thio-b-D-galactopyranoside at 37 C for 4 h. GNA1 was purified to homogeneity by nickel affinity chromatography and stored in buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 10 mM MgCl2, 10% glycerol) at 80 C. Typically 1–2 mg GNA1 could be obtained from 1 liter of cell culture. GNA1 activity assay GNA1 activity was measured as previously described with modifications [12]. The reaction mixture contained 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM GlcN6P, 0.5 mM Ac-CoA, and 0.5 mM DTNB in a final volume of 200 lL. Reaction was initiated by addition of GNA1 (0.01 to 0.1 lg) and monitored continuously at 412 nm at 37 C by a Uvikon XL UV/Vis spectrophotometer (Secomam, France). A blank reaction without Ac-CoA was set up as background control. The amount of CoA produced was calculated using the extinction coefficient of TNB at 37 C (13,800 M1 cm1) [17]. The specific activity of GNA1 was determined to be 200 units/mg protein. One enzyme unit is defined as one lmol CoA produced/min under the conditions specified above. Continuous assay by measuring Ac-CoA depletion In a quartz cuvette with 1 cm of optical path, the assay solution contained 50 mM Tris–HCl, Fru-6P (0.1– 6.0 mM), L-Gln (0.1–5.0 mM), 0.1 mM Ac-CoA, 2 lg GNA1 (400 units) in a final volume of 200 lL. The mixture was prewarmed at 37 C for 4 min. The reaction was initiated by adding GlmS (0.2–1.0 lg) and the disappearance of thioester bond at 230 nm was followed on an Uvikon xL UV/Vis spectrophotometer (Secomam, France) at 37 C. A blank reaction without GlmS was set up as background control. Kinetic parameters of GlmS were determined from initial velocity measurement at various concentrations of one substrate while keeping the other saturating. The extinction coefficient (e230 = 6436 M1 cm1) of Ac-CoA was determined experimentally. Discontinuous enzyme assay using Ellman’s reagent
Fig. 1. Schematic representation of the principle of the GNA1-coupled assay.
GlmS stock solution was changed to the following buffer containing no DTT (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10% glycerol) by passing through a PD-10 column (Amersham Bioscience). GlmS was further diluted into the same buffer containing 0.1 mg/mL bovine serum albumin to the necessary concentration before use. The following stock solutions were prepared: 0.5 M Tris–HCl (pH 7.5), 0.5 M KCl, 10 mM EDTA (pH 7.5), 60 mM Fru-6P, 50 mM L-Gln, 10 mM Ac-CoA. Ellman’s reagent solution contained 2 mM DTNB, 1 mM EDTA in buffer containing 50 mM Tris–HCl (pH 7.5). It was freshly prepared before use.
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Coupled assay for glucosamine-6P quantification / Y. Li et al. / Anal. Biochem. 370 (2007) 142–146
Enzymatic assays were performed in a 96-well PCR plate (Abgene) using a PCR heating block (Mastercycler gradient, Eppendorf). The total reaction volume was 50 lL. All stock solutions were prewarmed at 37 C. For measuring GlmS activity, 50 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 0.4 mM Ac–CoA, 6 mM Fru6P, 1 lg GNA1 (200 units), GlmS (0.1–0.6 lg) were well mixed. A blank reaction without Fru-6P was set up as reference. The reaction was initiated by adding 10 lL prewarmed L-Gln stock solution to the final concentration of 10 mM. The plate was capped with adhesive PCR film and incubated in the PCR heating block at 37 C for 5 min. The reaction was stopped by heating at 80 C for 4 min. The plate was left to cool for 5 min at room temperature before addition of 50 lL of Ellman’s reagent solution to each well. The mixture was incubated at room temperature for 10 min and 90 lL was transferred to a 96-well flatbottomed polystyrene plate. The absorbance at 412 nm was measured by a SpectraMax plus 384 UV reader (Molecular Devices). All readings were corrected for background in blank reaction lacking Fru-6P. A series of standards were set up in parallel with the enzyme reactions and subjected to the same process. Standards contained 2, 5, 10, 20, or 40 nmol CoASH in 50 mM Tris–HCl (pH 7.5) in a final volume of 50 lL. A reference without CoASH was used for background correction. To determine the Ki of inhibitors, enzyme reactions were carried out in the presence of inhibitor at different concentrations, while varying the amount of Fru-6P (0.1 to 4 mM) and keeping L-Gln at saturating concentration (10 mM). The kinetic data were analyzed by Igor Pro software (Oregon, USA). Results and discussion Continuous assay Inspired by the assay for pimeloyl-CoA synthase [18], the disappearance of Ac-CoA was followed spectrophotometrically at 230 nm which is specific for thioester bonds [14]. The initial rate of GlmS reaction was calculated from the slope A230 = f(t) during the first 1 or 2 min under these conditions. The hydrolysis of Ac-CoA was negligible and the production of GlcN6P was linear with GlmS quantity (0.2–1 lg). Interference at 230 nm by buffer components was corrected by running a control reaction lacking Ac-CoA before measurement. To demonstrate the utility of this method, the Km(Gln) and Km(Fru-6P) of GlmS from E. coli were determined to be 0.20 ± 0.02 and 0.25 ± 0.03 mM (Fig. 2), respectively, which are in good agreement with the values determined by other methods [19]. This is the first method reported so far that allows monitoring GlcN6P formation continuously. Compared to the conventionally used Morgan–Elson method, which requires several reagent additions and heating steps, the GNA1coupled continuous assay is particularly advantageous for determining kinetic parameters. However, the utility of this
Fig. 2. Representative determination of Km(L-Gln) for GlmS from E. coli. Continuous GNA1-coupled assay was performed at 37 C following the disappearance at 230 nm spectrophotometrically. Reactions contained 0.93 lg GlmS, 6 mM Fru-6P, various concentrations of L-Gln (0.2 to 6 mM), 0.1 mM Ac-CoA, 2 lg (400 units) GNA1 in assay buffer in a total volume of 200 lL. Km(Gln) was determined to be 0.20 ± 0.02 mM; Vmax was 7.2 ± 0.3 units/mg protein. Error bars represent standard deviations.
continuous assay in the 96-well format is hampered by the availability of expensive quartz microtiter plates. Moreover, for inhibitor screening, it is more susceptible to interference since many compounds have absorption at 230 nm. Therefore an alternative detection method was developed to circumvent these inconveniences for inhibitor screening. Discontinuous assay using Ellman’s reagent The activity of GNA1 can be measured continuously in the presence of Ellman reagent (DTNB) at 420 nm. However, considering that GlmS requires Cys1 residue for catalytic function and thus is susceptible to any thiolmodifying reagents [1], a discontinuous GNA1-coupled assay was developed. Enzyme reactions were carried out in a 96-well PCR plate as described for the MS-based assay for GlmS [10]. Utilization of a PCR heating block facilitates inactivation of the enzymes by a temperature jump to 80 C, avoiding addition of guanidine or urea solution. In the assay system, GNA1 was kept in large excess (1– 2 lg, 200–400 units) in order to reflect the true velocity of GlmS reaction. Ac-CoA was fixed to 0.4 mM (2 · Km) for GNA1 [12]. Nonenzymatic hydrolysis of Ac-CoA (about 2.7%) under assay conditions was the contribution to background (A412 0.03), which can be corrected by setting up a blank control lacking one substrate of GlmS. Reducing reagents such as DTT in the enzyme preparation interfere with the DTNB reaction, therefore GlmS was freshly desalted by gel filtration before each assay. Quantification was carried out according to a standard curve. A series of standards which contained known quantities of CoASH in the reaction buffer was set up and subjected to the same process as enzyme reactions. This ensures batch-to-batch reproducibility and reliable calculations. Using a known quantity of GlcN6P, it was confirmed that equal amounts of CoASH were produced
Coupled assay for glucosamine-6P quantification / Y. Li et al. / Anal. Biochem. 370 (2007) 142–146
Fig. 3. (a) GlcN6P production is linear with enzyme quantity. (b) GlcN6P production is linear with the incubation time (0.12 lg GlmS). The reaction was carried out at 37 C for 5 min with 6 mM Fru-6P, 10 mM L-Gln, 0.8 mM Ac-CoA under conditions specified under materials and methods. The amount of GlcN6P was deduced from the amount of CoASH produced. Error bars represent standard deviations.
by GNA1 under the assay conditions. The linear range of the standard curve is 2 to 50 nmol (final concentration: 0.02–0.5 mM). The production of GlcN6P was proportional to enzyme quantity (0.1–0.6 lg, Fig. 3a) and assay
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duration under the condition specified (Fig. 3b). To validate the method, the inhibition constants of two inhibitors of GlmS from E. coli were determined (Fig. 4). The inhibitors were first tested to verify the absence of inhibition of GNA1 itself. 2-Amino-2-deoxy-D-glucitol-6-phosphate has been reported as a known competitive inhibitor of the Fru6P binding site. The determined Ki of 18 ± 3 lM is in excellent agreement with the previous reported value (20 lM) [20], demonstrating that this method is suitable for inhibitor studies (Fig. 4b). We further determined the Ki of 2-amino-2-deoxy-D-mannitol-6-phosphate (ADMP), a previous uncharacterized inhibitor of GlmS from E. coli, to be 0.67 ± 0.13 lM. The Ki of ADMP on GlmS from Candida albicans was determined to be 9 lM by a modified Elson– Morgan method in a recent study [21]. The difference between the two Ki values may be an inherent property of GlmS from two different organisms. Nevertheless, our result confirms that ADMP is the strongest inhibitor of the Fru-6P site of GlmS so far developed. Compared to other methods of GlcN6P detection, the GNA1-coupled discontinuous assay is simple and convenient to perform. The sensitivity is within the range of other UV-based methods. The reliable detection limit (2 nmol) is 5 and 10 times lower than that of the MS-based and colorimetric Morgan–Elson methods, respectively. Provided that an instrument which automates temperature control, reagent addition and UV reading is available, high-throughput screening of GlmS inhibitors by this method is feasible. Moreover, recent advances in discovering fluorescent probes for thiol quantification make it promising to ameliorate the sensitivity of the GNA1-coupled assay down to the picomolar range, which would be comparable with the radioactive assay. Worthy of note, in an attempt to use the water-soluble fluorescent thiol probes developed by Maeda et al. [22], it was observed that GNA1 hydrolyses the probe, giving unacceptable back-
Fig. 4. Representative determination of Ki of 2-amino-2-deoxy-D-glucitol-6-phosphate. Discontinuous assay using Ellman’s reagent was carried out to determine Ki. GlmS (0.5 lg) was incubated with Fru-6P of various concentrations (0.1 to 4 mM) in the presence of the inhibitor (m 0 lM, s 10 lM, h 20 lM, j 50 lM, d 100 lM, 150 lM). L-Gln, Ac-CoA, and GNA1 were fixed at 10 mM, 0.4 mM, and 1 lg, respectively, in a total volume of 50 lL. The data represent the mean of triplicates. (a) Hanes–Woolf presentation of data. (b) Ki (18 lM) was determined from intercepts. Km(app): apparent Km.
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Coupled assay for glucosamine-6P quantification / Y. Li et al. / Anal. Biochem. 370 (2007) 142–146
ground even with traces of remaining GNA1 activity, which attenuates the application of these probes to our assay. Finally, it should be mentioned that the GNA1-coupled method is more adapted for measuring activity with purified enzyme, as the presence of Ac-CoA hydrolase in tissue samples may interfere with the assay. Obviously, for inhibitor screening, it must be verified that the positive hits neither inhibit GNA1 nor react with Ellman’s reagent. In summary, an enzyme-coupled assay for measuring the amidotransferase activity of GlmS was developed. The two formats of detection, either discontinuous or continuous, may be chosen as needed. Its simplicity and reliability are suitable for high-throughput screenings and enzymatic studies. Acknowledgments We thank Professor H. Maeda (Osaka University, Japan) for kind gifts of fluorescent thiol probes and Drs. O. Ploux and S. Mann (University of Paris VI) for advice on the continuous assay. Y. Li was supported by an ICSN postdoctoral fellowship. The project was supported by IFCPAR-Project No. 3003-1. References [1] A. Teplyakov, C. Leriche, G. Obmolova, B. Badet, M.A. BadetDenisot, From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase, Nat. Prod. Rep. 19 (2002) 60–69. [2] E. Borowski, Novel approaches in the rational design of antifungal agents of low toxicity, Farmaco 55 (2000) 206–208. [3] M.G. Buse, Hexosamines, insulin resistance, and the complications of diabetes: current status, Am. J. Physiol. Endocrinol. Metab. 290 (2006) E1–E8. [4] F. Massie`re, M.A. Badet-Denisot, The mechanism of glutaminedependent amidotransferases, Cell. Mol. Life Sci. 54 (1998) 205–222. [5] A. Teplyakov, G. Obmolova, B. Badet, M.A. Badet-Denisot, Channeling of ammonia in glucosamine-6-phosphate synthase, J. Mol. Biol. 313 (2001) 1093–1102. [6] K.O. Broschat, C. Gorka, J.D. Page, C.L. Martin-Berger, M.S. Davies, H. Huang, E.A. Gulve, W.J. Salsgiver, T.P. Kasten, Kinetic characterization of human glutamine-fructose-6-phosphate amdotransferase 1, J. Biol. Chem. 277 (2002) 14764–14770. [7] S. Ghosh, H.J. Blumenthal, E. Davidson, S. Roseman, Glucosamine metabolism V. Enzymatic synthesis of glucosamine-6-phosphate, J. Biol. Chem. 235 (1960) 1265–1273.
[8] C. Burghardt, and J. P. Kochan, Method for measuring glucosamine6-phosphate, European Patent 1431396A1, 2004. [9] M.C. Daniels, T.P. Ciaraldi, S. Nikoulina, R.R. Henry, D.A. McClain, Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells, J. Clin. Invest. 97 (1996) 1235–1241. [10] L.T. Maillard, V. Gue´rineau, M.A. Badet-Denisot, B. Badet, O. Lapre´vote, P. Durand, Monitoring enzyme-catalyzed production of glucosamine-6P by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry: a new enzymatic assay for glucosamine-6P synthase, Rapid Commun. Mass Spectrom. 20 (2006) 666– 672. [11] K.O. Broschat, C. Gorka, T.P. Kasten, E.A. Gulve, B. Kilpatrick, A radiometric assay for glutamine: fructose-6-phosphate amidotransferase, Anal. Biochem. 305 (2002) 10–15. [12] T. Mio, T. Yamada-Okabe, M. Arisawa, H. Yamada-Okabe, Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis, J. Biol. Chem. 274 (1999) 424–429. [13] G.L. Ellman, A colorimetric method for determining low concentrations of mercaptans, Arch. Biochem. Biophys. 82 (1959) 70–77. [14] E.R. Stadtman, Preparation and assay of acyl coenzyme A and other thiol esters; use of hydroxylamine, Methods Enzymol. 3 (1957) 931– 941. [15] S. Mouilleron, M.A. Badet-Denisot, B. Golinelli-Pimpaneau, Glutamine binding opens the ammonia channel and activates glucosamine6P synthase, J. Biol. Chem. 281 (2006) 4404–4412. [16] C. Peneff, D. Mengin-Lecreulx, Y. Bourne, The crystal structures of apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase, J. Biol. Chem. 276 (2001) 16328–16334. [17] P. Eyer, F. Worek, D. Kiderien, G. Sinko, A. Stuglin, V. SimeonRudolf, E. Reiner, Molar absorption coefficients for the reduced Ellman reagent: reassessment, Anal. Biochem. 312 (2003) 224–227. [18] O. Ploux, P. Soularue, A. Marquet, R. Gloeckler, Y. Lemoine, Investigation of the first step of biotin biosynthesis in Bacillus sphaericus, Biochem. J. 287 (1992) 685–690. [19] B. Badet, P. Vermoote, P.Y. Haumont, F. Lederer, F. LeGoffic, Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location, Biochemistry 26 (1987) 1940–1948. [20] M.A. Badet-Denisot, C. Leriche, F. Masserie, B. Badet, Nitrogen transfer in E. coli glucosamine-6-phosphate synthase: Investigation using substrate and bisubstrate analogs, Bioorg. Med. Chem. Lett. 5 (1995) 815–820. [21] S. Milewski, A. Janiak, M. Wojciechowski, Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate syntase and phosphoglucose isomerase, Arch. Biochem. Biophys. 450 (2006) 39–49. [22] H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki, N. Itoh, 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman’s reagent in thiol-quantification enzyme assays, Angew. Chem. Int. Ed. 44 (2005) 2922–2925.
An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase Yanyan Li, Philippe Lopez, Philippe Durand, Jamal Ouazzani, Bernard Badet, Marie-Ange Badet-Denisot * Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-Sur-Yvette, France Received 12 April 2007 Available online 9 August 2007
Abstract An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-coenzyme A (CoA) to glucosamine-6-phosphate, releasing coenzyme A. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,5 0 -dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS. 2007 Elsevier Inc. All rights reserved. Keywords: Glucosamine-6-phosphate synthase; Acetyltransferase; Ellman’s reagent; Enzymatic test
Hexosamine biosynthesis is ubiquitous in all organisms and the end products of this pathway are essential sugar building blocks for biosynthesis of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals [1]. The first and rate-limiting reaction is catalyzed by glucosamine6-phosphate synthase (GlmS, EC 2.6.1.16),1 which converts D-fructose-6-phosphate (Fru-6P) into D-glucosamine-6-phosphate (GlcN6P) with hydrolysis of L-glutamine to L-glutamate. As an important enzyme controlling the amino sugar metabolism, GlmS is proposed to be a thera-
*
Corresponding author. Fax: +33 1 69077247. E-mail address: [email protected] (M.-A. Badet-Denisot). 1 Abbreviations used: Ac, acetyl; ADGP, 2-amino-2-deoxy-D-glucitol-6phosphate; ADMP, 2-amino-2-deoxy-D-mannitol-6-phosphate; DTNB, 5,5 0 -dithio-bis(2-nitrobenzoic acid); Fru-6P, D-fructose-6-phosphate; GlmS, bacterial glucosamine-6-phosphate synthase; GlcN6P, glucosamine-6-phosphate; GNA1, yeast glucosamine-6-phosphate N-acetyltransferase 1; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; TNB, 2-nitrobenzoic acid; DTT, dithiothreitol. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.07.031
peutic target in the development of antifungal [2], and more recently, antidiabetic compounds [3]. GlmS belongs to the N-terminal nucleophilic glutaminedependent amidotransferase family [4]. Structural studies have shown that GlmS from Escherichia coli possesses an intramolecular tunnel through which ammonia is transferred from the glutamine site to the Fru-6P binding site for amination [5]. Detailed investigations on the functioning of the tunnel, such as its blockage or perforation, require in particular knowledge of the amidotransferase activity (GlcN6P synthesizing activity) and thus reliable quantification of GlcN6P. Enzyme activity of GlmS was conveniently measured by quantification of glutamate using glutamate dehydrogenase as coupling enzyme. However, this method has limits in screening inhibitors of the Fru-6P binding site, since glutamate production is not always coupled to amidotransferase activity [6]. Several methods were developed for monitoring GlcN6P formation, including a colorimetric Morgan– Elson assay [7], which was adapted to microtiter plate format for inhibitor screening [8], a fluorometric assay with o-phthaldialdehyde derivatization [9], and a MALDI-TOF
Coupled assay for glucosamine-6P quantification / Y. Li et al. / Anal. Biochem. 370 (2007) 142–146
mass spectrometry-based assay [10]. They are all noncontinuous assays and require multiple derivatization steps. Recently a radiometric assay using 14C-radiolabeled Fru6P, which allows sensitive detection of GlcN6P down to 1 pmol and can be adapted to 96-well plate format, was developed [11]. However handling of radioactivity requires special caution. Here we report a simple and rapid assay for GlcN6P detection using yeast GlcN6P N-acetyltransferase 1 (GNA1) as coupling enzyme (Fig. 1). In eukaryotes, GNA1 transfers the acetyl moiety from acetyl-CoA (AcCoA) to GlcN6P, producing N-acetyl-GlcN6P and coenzyme A (CoASH) [12]. The free sulfhydryl group of CoASH can be then quantified by the widely used Ellman’s reagent, 5,5 0 -dithio-bis-(2-nitrobenzoic acid) (DTNB) in a discontinuous manner [13]. Alternatively, GlcN6P formation is followed by the disappearance of the thioester bond of Ac-CoA at 230 nm [14]. This is the first continuous method that allows monitoring GlcN6P production directly. Validation of the assay by determining the kinetic parameters of GlmS from E. coli and inhibition constants (Ki) of two inhibitors is presented in this report. Materials and methods General materials Chemicals were purchased from Sigma Chemical Co. and were of highest purity available. L-Gln was further purified by recrystallization. The 96-well PCR plates (Thermo-Fast 96, nonskirted) and adhesive PCR films were from Abgene (UK). The 96-well flat-bottomed polystyrene plates for UV measurement were from Becton–Dickinson Labware (USA). GlmS was purified and stored in buffer (50 mM Bis-Tris-propane, pH 7.2, 1 mM DTT, 1 mM EDTA, 10% glycerol) at 80 C as previously described [15]. Overexpression and purification of GNA1 Plasmid pET28-gna1 for expressing yeast GNA1 with a His6 tag was a kind gift of Dr. D. Mengin-Lecreulx (Universite´ Paris-Sud, Orsay, France) [16]. E. coli BL21(DE3)-
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pLyS cells harboring pET28-gna1 were grown at 37 C with agitation until OD600 reached 0.7–1. Protein expression was induced with 0.2 mM isopropyl-L-thio-b-D-galactopyranoside at 37 C for 4 h. GNA1 was purified to homogeneity by nickel affinity chromatography and stored in buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 10 mM MgCl2, 10% glycerol) at 80 C. Typically 1–2 mg GNA1 could be obtained from 1 liter of cell culture. GNA1 activity assay GNA1 activity was measured as previously described with modifications [12]. The reaction mixture contained 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM GlcN6P, 0.5 mM Ac-CoA, and 0.5 mM DTNB in a final volume of 200 lL. Reaction was initiated by addition of GNA1 (0.01 to 0.1 lg) and monitored continuously at 412 nm at 37 C by a Uvikon XL UV/Vis spectrophotometer (Secomam, France). A blank reaction without Ac-CoA was set up as background control. The amount of CoA produced was calculated using the extinction coefficient of TNB at 37 C (13,800 M1 cm1) [17]. The specific activity of GNA1 was determined to be 200 units/mg protein. One enzyme unit is defined as one lmol CoA produced/min under the conditions specified above. Continuous assay by measuring Ac-CoA depletion In a quartz cuvette with 1 cm of optical path, the assay solution contained 50 mM Tris–HCl, Fru-6P (0.1– 6.0 mM), L-Gln (0.1–5.0 mM), 0.1 mM Ac-CoA, 2 lg GNA1 (400 units) in a final volume of 200 lL. The mixture was prewarmed at 37 C for 4 min. The reaction was initiated by adding GlmS (0.2–1.0 lg) and the disappearance of thioester bond at 230 nm was followed on an Uvikon xL UV/Vis spectrophotometer (Secomam, France) at 37 C. A blank reaction without GlmS was set up as background control. Kinetic parameters of GlmS were determined from initial velocity measurement at various concentrations of one substrate while keeping the other saturating. The extinction coefficient (e230 = 6436 M1 cm1) of Ac-CoA was determined experimentally. Discontinuous enzyme assay using Ellman’s reagent
Fig. 1. Schematic representation of the principle of the GNA1-coupled assay.
GlmS stock solution was changed to the following buffer containing no DTT (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10% glycerol) by passing through a PD-10 column (Amersham Bioscience). GlmS was further diluted into the same buffer containing 0.1 mg/mL bovine serum albumin to the necessary concentration before use. The following stock solutions were prepared: 0.5 M Tris–HCl (pH 7.5), 0.5 M KCl, 10 mM EDTA (pH 7.5), 60 mM Fru-6P, 50 mM L-Gln, 10 mM Ac-CoA. Ellman’s reagent solution contained 2 mM DTNB, 1 mM EDTA in buffer containing 50 mM Tris–HCl (pH 7.5). It was freshly prepared before use.
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Enzymatic assays were performed in a 96-well PCR plate (Abgene) using a PCR heating block (Mastercycler gradient, Eppendorf). The total reaction volume was 50 lL. All stock solutions were prewarmed at 37 C. For measuring GlmS activity, 50 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 0.4 mM Ac–CoA, 6 mM Fru6P, 1 lg GNA1 (200 units), GlmS (0.1–0.6 lg) were well mixed. A blank reaction without Fru-6P was set up as reference. The reaction was initiated by adding 10 lL prewarmed L-Gln stock solution to the final concentration of 10 mM. The plate was capped with adhesive PCR film and incubated in the PCR heating block at 37 C for 5 min. The reaction was stopped by heating at 80 C for 4 min. The plate was left to cool for 5 min at room temperature before addition of 50 lL of Ellman’s reagent solution to each well. The mixture was incubated at room temperature for 10 min and 90 lL was transferred to a 96-well flatbottomed polystyrene plate. The absorbance at 412 nm was measured by a SpectraMax plus 384 UV reader (Molecular Devices). All readings were corrected for background in blank reaction lacking Fru-6P. A series of standards were set up in parallel with the enzyme reactions and subjected to the same process. Standards contained 2, 5, 10, 20, or 40 nmol CoASH in 50 mM Tris–HCl (pH 7.5) in a final volume of 50 lL. A reference without CoASH was used for background correction. To determine the Ki of inhibitors, enzyme reactions were carried out in the presence of inhibitor at different concentrations, while varying the amount of Fru-6P (0.1 to 4 mM) and keeping L-Gln at saturating concentration (10 mM). The kinetic data were analyzed by Igor Pro software (Oregon, USA). Results and discussion Continuous assay Inspired by the assay for pimeloyl-CoA synthase [18], the disappearance of Ac-CoA was followed spectrophotometrically at 230 nm which is specific for thioester bonds [14]. The initial rate of GlmS reaction was calculated from the slope A230 = f(t) during the first 1 or 2 min under these conditions. The hydrolysis of Ac-CoA was negligible and the production of GlcN6P was linear with GlmS quantity (0.2–1 lg). Interference at 230 nm by buffer components was corrected by running a control reaction lacking Ac-CoA before measurement. To demonstrate the utility of this method, the Km(Gln) and Km(Fru-6P) of GlmS from E. coli were determined to be 0.20 ± 0.02 and 0.25 ± 0.03 mM (Fig. 2), respectively, which are in good agreement with the values determined by other methods [19]. This is the first method reported so far that allows monitoring GlcN6P formation continuously. Compared to the conventionally used Morgan–Elson method, which requires several reagent additions and heating steps, the GNA1coupled continuous assay is particularly advantageous for determining kinetic parameters. However, the utility of this
Fig. 2. Representative determination of Km(L-Gln) for GlmS from E. coli. Continuous GNA1-coupled assay was performed at 37 C following the disappearance at 230 nm spectrophotometrically. Reactions contained 0.93 lg GlmS, 6 mM Fru-6P, various concentrations of L-Gln (0.2 to 6 mM), 0.1 mM Ac-CoA, 2 lg (400 units) GNA1 in assay buffer in a total volume of 200 lL. Km(Gln) was determined to be 0.20 ± 0.02 mM; Vmax was 7.2 ± 0.3 units/mg protein. Error bars represent standard deviations.
continuous assay in the 96-well format is hampered by the availability of expensive quartz microtiter plates. Moreover, for inhibitor screening, it is more susceptible to interference since many compounds have absorption at 230 nm. Therefore an alternative detection method was developed to circumvent these inconveniences for inhibitor screening. Discontinuous assay using Ellman’s reagent The activity of GNA1 can be measured continuously in the presence of Ellman reagent (DTNB) at 420 nm. However, considering that GlmS requires Cys1 residue for catalytic function and thus is susceptible to any thiolmodifying reagents [1], a discontinuous GNA1-coupled assay was developed. Enzyme reactions were carried out in a 96-well PCR plate as described for the MS-based assay for GlmS [10]. Utilization of a PCR heating block facilitates inactivation of the enzymes by a temperature jump to 80 C, avoiding addition of guanidine or urea solution. In the assay system, GNA1 was kept in large excess (1– 2 lg, 200–400 units) in order to reflect the true velocity of GlmS reaction. Ac-CoA was fixed to 0.4 mM (2 · Km) for GNA1 [12]. Nonenzymatic hydrolysis of Ac-CoA (about 2.7%) under assay conditions was the contribution to background (A412 0.03), which can be corrected by setting up a blank control lacking one substrate of GlmS. Reducing reagents such as DTT in the enzyme preparation interfere with the DTNB reaction, therefore GlmS was freshly desalted by gel filtration before each assay. Quantification was carried out according to a standard curve. A series of standards which contained known quantities of CoASH in the reaction buffer was set up and subjected to the same process as enzyme reactions. This ensures batch-to-batch reproducibility and reliable calculations. Using a known quantity of GlcN6P, it was confirmed that equal amounts of CoASH were produced
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Fig. 3. (a) GlcN6P production is linear with enzyme quantity. (b) GlcN6P production is linear with the incubation time (0.12 lg GlmS). The reaction was carried out at 37 C for 5 min with 6 mM Fru-6P, 10 mM L-Gln, 0.8 mM Ac-CoA under conditions specified under materials and methods. The amount of GlcN6P was deduced from the amount of CoASH produced. Error bars represent standard deviations.
by GNA1 under the assay conditions. The linear range of the standard curve is 2 to 50 nmol (final concentration: 0.02–0.5 mM). The production of GlcN6P was proportional to enzyme quantity (0.1–0.6 lg, Fig. 3a) and assay
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duration under the condition specified (Fig. 3b). To validate the method, the inhibition constants of two inhibitors of GlmS from E. coli were determined (Fig. 4). The inhibitors were first tested to verify the absence of inhibition of GNA1 itself. 2-Amino-2-deoxy-D-glucitol-6-phosphate has been reported as a known competitive inhibitor of the Fru6P binding site. The determined Ki of 18 ± 3 lM is in excellent agreement with the previous reported value (20 lM) [20], demonstrating that this method is suitable for inhibitor studies (Fig. 4b). We further determined the Ki of 2-amino-2-deoxy-D-mannitol-6-phosphate (ADMP), a previous uncharacterized inhibitor of GlmS from E. coli, to be 0.67 ± 0.13 lM. The Ki of ADMP on GlmS from Candida albicans was determined to be 9 lM by a modified Elson– Morgan method in a recent study [21]. The difference between the two Ki values may be an inherent property of GlmS from two different organisms. Nevertheless, our result confirms that ADMP is the strongest inhibitor of the Fru-6P site of GlmS so far developed. Compared to other methods of GlcN6P detection, the GNA1-coupled discontinuous assay is simple and convenient to perform. The sensitivity is within the range of other UV-based methods. The reliable detection limit (2 nmol) is 5 and 10 times lower than that of the MS-based and colorimetric Morgan–Elson methods, respectively. Provided that an instrument which automates temperature control, reagent addition and UV reading is available, high-throughput screening of GlmS inhibitors by this method is feasible. Moreover, recent advances in discovering fluorescent probes for thiol quantification make it promising to ameliorate the sensitivity of the GNA1-coupled assay down to the picomolar range, which would be comparable with the radioactive assay. Worthy of note, in an attempt to use the water-soluble fluorescent thiol probes developed by Maeda et al. [22], it was observed that GNA1 hydrolyses the probe, giving unacceptable back-
Fig. 4. Representative determination of Ki of 2-amino-2-deoxy-D-glucitol-6-phosphate. Discontinuous assay using Ellman’s reagent was carried out to determine Ki. GlmS (0.5 lg) was incubated with Fru-6P of various concentrations (0.1 to 4 mM) in the presence of the inhibitor (m 0 lM, s 10 lM, h 20 lM, j 50 lM, d 100 lM, 150 lM). L-Gln, Ac-CoA, and GNA1 were fixed at 10 mM, 0.4 mM, and 1 lg, respectively, in a total volume of 50 lL. The data represent the mean of triplicates. (a) Hanes–Woolf presentation of data. (b) Ki (18 lM) was determined from intercepts. Km(app): apparent Km.
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ground even with traces of remaining GNA1 activity, which attenuates the application of these probes to our assay. Finally, it should be mentioned that the GNA1-coupled method is more adapted for measuring activity with purified enzyme, as the presence of Ac-CoA hydrolase in tissue samples may interfere with the assay. Obviously, for inhibitor screening, it must be verified that the positive hits neither inhibit GNA1 nor react with Ellman’s reagent. In summary, an enzyme-coupled assay for measuring the amidotransferase activity of GlmS was developed. The two formats of detection, either discontinuous or continuous, may be chosen as needed. Its simplicity and reliability are suitable for high-throughput screenings and enzymatic studies. Acknowledgments We thank Professor H. Maeda (Osaka University, Japan) for kind gifts of fluorescent thiol probes and Drs. O. Ploux and S. Mann (University of Paris VI) for advice on the continuous assay. Y. Li was supported by an ICSN postdoctoral fellowship. The project was supported by IFCPAR-Project No. 3003-1. References [1] A. Teplyakov, C. Leriche, G. Obmolova, B. Badet, M.A. BadetDenisot, From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase, Nat. Prod. Rep. 19 (2002) 60–69. [2] E. Borowski, Novel approaches in the rational design of antifungal agents of low toxicity, Farmaco 55 (2000) 206–208. [3] M.G. Buse, Hexosamines, insulin resistance, and the complications of diabetes: current status, Am. J. Physiol. Endocrinol. Metab. 290 (2006) E1–E8. [4] F. Massie`re, M.A. Badet-Denisot, The mechanism of glutaminedependent amidotransferases, Cell. Mol. Life Sci. 54 (1998) 205–222. [5] A. Teplyakov, G. Obmolova, B. Badet, M.A. Badet-Denisot, Channeling of ammonia in glucosamine-6-phosphate synthase, J. Mol. Biol. 313 (2001) 1093–1102. [6] K.O. Broschat, C. Gorka, J.D. Page, C.L. Martin-Berger, M.S. Davies, H. Huang, E.A. Gulve, W.J. Salsgiver, T.P. Kasten, Kinetic characterization of human glutamine-fructose-6-phosphate amdotransferase 1, J. Biol. Chem. 277 (2002) 14764–14770. [7] S. Ghosh, H.J. Blumenthal, E. Davidson, S. Roseman, Glucosamine metabolism V. Enzymatic synthesis of glucosamine-6-phosphate, J. Biol. Chem. 235 (1960) 1265–1273.
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