ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 360 (2007) 227–234

www.elsevier.com/locate/yabio

A rapid assay for dihydropteroate synthase activity suitable for identiWcation of inhibitors

Ross T. Fernley ¤, Peter Iliades 1, Ian Macreadie

CSIRO, Molecular and Health Technologies, Parkville, Vic. 3052, Australia

Received 27 June 2006Available online 13 November 2006

Abstract

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps infolate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimi-crobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugsis increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable toautomation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihy-dropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation ofNADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measurekinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and repro-ducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could beused for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the Wrst step in developing new antimicrobialdrugs targeting the folate biosynthetic pathway.Crown copyright © 2006 Published by Elsevier Inc. All rights reserved.

Keywords: Folate; Sulfa drugs; Dihydropteroate synthase; Dihydrofolate reductase; Hydroxymethyldihydropterin pyrophosphokinase; Spectrophoto-metric; Continuous assay

Folate cofactors are essential components of several makes the enzymes of this pathway ideal targets for antimi-

enzymatic reactions occurring in nearly all cells and areessential for life. They are involved in the biosynthesis ofpurines and pyrimidines (thymidylate), the biosynthesis ofamino acids such as glycine and methionine, and the bio-synthesis of vitamins such as pantothenic acid [1]. The reac-tions involve the transfer of one-carbon units to and fromfolate cofactors. Microorganisms and plants are able tosynthesize folates in a pathway involving six enzymes, start-ing with GTP cyclohydrolase (EC 3.5.4.16) [2]. Mammalslack this pathway and must obtain folates from the diet in aprocess involving speciWc folate uptake receptors [3]. Theabsence of a folate biosynthetic pathway in mammals

* Corresponding author. Fax: +61 3 9662 7101.E-mail address: ross.fernley@csiro.au (R.T. Fernley).

1 Present address: Walter and Eliza Hall Institute of Medical Research,Melbourne 3050, Australia.

0003-2697/$ - see front matter Crown copyright © 2006 Published by Elsevierdoi:10.1016/j.ab.2006.10.036

crobial drugs. Indeed, the Wrst chemically synthesized anti-microbials—the sulfonamides or sulfa drugs—targeted oneof these enzymes, namely dihydropteroate synthase(DHPS2, EC 2.5.1.15) [4]. These sulfa drugs are still widelyused, usually in combination with dihydrofolate reductase(DHFR, EC 1.5.1.3) inhibitors, to treat bacterial infections(sulfamethoxazole), malaria (sulfadoxine), and Pneumocys-tis pneumonia (sulfamethoxazole). Another sulfa drug,

2 Abbreviations used: DHPS, 7,8-dihydropteroate synthase; DHFR,dihydrofolate reductase; HPPK, 6-hydroxymethyl-7,8-dihydropterin py-rophosphokinase; PCP, P. carinii pneumonia; DHNA, dihydroneopterinaldolase; cDNA, complementary DNA; NCBI, National Center for Bio-technology Information; p-ABA, p-aminobenzoic acid; sulfamethoxazole,4-amino-N-(5-methyl-3-isoxazolyl)benzene sulfonamide; dapsone, 4-amin-ophenylsulfone; sulfachloropyridazine, 4-amino-N-(6-chloro-3-pyridazi-nyl)benzene sulfonamide; DMSO, dimethyl sulfoxide; NMR, nuclearmagnetic resonance.

Inc. All rights reserved.

228 Rapid assay for dihydropteroate synthase / R.T. Fernley et al. / Anal. Biochem. 360 (2007) 227–234

dapsone, is used to treat leprosy in combination with otherdrugs. Together, these combinations work synergisticallyand slow the development of drug resistance signiWcantly.However, resistance to these drugs evolves and is increasing[5]. Thus, there is an urgent need to develop new drugs tocombat these widespread diseases.

In prokaryotes the folate biosynthetic enzymes areexpressed as individual monofunctional proteins, whereasin some eukaryotic microorganisms these enzymes oftenare expressed as multifunctional enzymes. For example, inPlasmodium falciparum, the causative agent for the mostserious form of malaria, two enzymes catalyzing sequentialsteps in folate biosynthesis occur as a bifunctional proteinwith 6-hydroxymethyl-7,8-dihydropterin pyrophosphokin-ase (HPPK, EC 2.7.6.3) and DHPS activities [6]. This is alsothe case for Toxoplasma gondii, a parasite causing opportu-nistic infection in immunocompromised patients [7]. Threesequential enzymes of this pathway occur as a trifunctionalprotein in the fungus Pneumocystis carinii [8], a cause ofpneumonia in immunocompromised patients (oftenreferred to as PCP [P. carinii pneumonia]). The yeast Sac-charomyces cerevisiae has a similar trifunctional enzyme [9].These proteins contain enzyme activities of dihydroneop-terin aldolase (DHNA, EC 4.1.2.25) as well as HPPK andDHPS activities.

Recently, we generated a complementary DNA (cDNA)encoding the HPPK and DHPS domains of the S. cerevi-siae multifunctional protein. This was overexpressed, puri-Wed, and crystallized [10], and its three-dimensionalstructure was determined in a complex with a substrateanalog [11]. Our aim is to develop new drugs that targetthese enzyme activities using structure-based drug design,incorporating in silico screening of chemical libraries fol-lowed by in vitro evaluation of potential inhibitors. Theevaluation of potential inhibitors discovered in the processrequires a robust rapid assay to test potentially a largenumber of chemicals. The method currently in use to mea-sure the activities of HPPK and DHPS involves incorpora-tion of radioactively labeled substrate into the pterinprecursor, separation of labeled substrate from product bythin-layer chromatography, and measurement of the incor-porated label [12,13]. This method is slow and tedious andis not suited to high-throughput screening necessary toidentify and characterize potential inhibitors. The alternatepatented method [14] uses pyrophosphatase to measurepyrophosphate released from the DHPS reaction. However,this is not a real-time method; rather, it requires that thereaction be stopped and that pyrophosphatases be added tomeasure pyrophosphate formed in the reaction well. Thus,this assay methodology necessitates multiple reactions atnumerous time points to achieve kinetic data.

This article describes a new method for assaying HPPKand DHPS activities that is rapid, is continuous, and can bereadily automated for rapid screening of chemical librariesfor potential inhibitors. To demonstrate its usefulness, thebinding constants for three sulfa drugs with wild-type andsulfa drug-resistant forms of the enzyme are determined here.

Materials and methods

Enzymes

The yeast bifunctional HPPK–DHPS enzyme wasexpressed in Escherichia coli and puriWed by metal aYnitychromatography and ion exchange chromatography asdescribed previously [10]. The His6 tag was removed bythrombin cleavage, and the enzyme was stored in 50% glyc-erol at ¡20 °C. The QuikChange XL site-directed mutagene-sis kit (Stratagene, La Jolla, CA, USA) was used to generatethe four mutant DHPS alleles from the HPPK–DHPS con-struct [15]. The wild-type yeast construct has the sequenceT557R558P559 (National Center for Biotechnology Informa-tion [NCBI] entry P53848), whereas the single mutant allelesare TRS559 (called TRS DHPS here) and A557RP (ARPDHPS). The double mutant alleles are V557RS559 (VRSDHPS) and A557RS559 (ARS DHPS) [16]. The numberingsystem for S. cerevisiae DHNA–HPPK–DHPS diVers fromthe earlier numbering of this protein in that the start site forthe gene has been moved 40 codons downstream [17]. TheE. coli BL21 (DE3) strain transformed separately with thefour N-terminal His6-tagged pET-28a.Sc HPPK–DHPS vec-tors was grown in 2£ YT medium supplemented with 40�g/ml kanamycin as described previously [10] for the wild-typeenzyme. The proteins were also puriWed as described previ-ously [10]. Protein concentrations were determined by theBradford protein assay using bovine serum albumin as stan-dard (Bio-Rad, Regents Park, Australia). Dihydrofolatereductase (bovine liver) was obtained from Sigma (St. Louis,MO, USA).

Reagents

6-Hydroxymethylpterin pyrophosphate (Schircks Labora-tories, Jona, Switzerland) was reduced with sodium dithio-nite–ascorbic acid to 6-hydroxymethyl-7,8-dihydropterinpyrophosphate and puriWed by ion exchange chromatogra-phy on Mono Q resin as described previously [10]. ThisDHPS substrate was stored in aliquots at ¡80°C. 6-Hydroxy-methyl-7,8-dihydropterin, 7,8-dihydropteroate, 7,8-dihydro-folate, and 5,6,7,8-tetrahydrofolate were obtained fromSchircks, and p-aminobenzoic acid (p-ABA) and NADPHwere obtained from Sigma.

4-Amino-N-(5-methyl-3-isoxazolyl)benzene sulfonamide(sulfamethoxazole, Sigma), 4-aminophenylsulfone (dapsone,Aldrich, Milwaukee, WI, USA), and 4-amino-N-(6-chloro-3-pyridazinyl)benzene sulfonamide (sulfachloropyridazine,ICN Biomedicals, Seven Hills, Australia) were dissolved indimethyl sulfoxide (DMSO, BDH AnalaR) to 100 mg/ml.Working dilutions were made in DMSO from these stocksolutions.

Assay

Initially, the assay was carried out in 0.6-ml quartz spec-trophotometric cells (Starna, Thornleigh, Australia).

Rapid assay for dihydropteroate synthase / R.T. Fernley et al. / Anal. Biochem. 360 (2007) 227–234 229

BuVers and reagents were equilibrated at the assay temper-ature, and the assay was started by the addition of HPPK–DHPS. The absorbance at 340 nm was monitored over3 min in a Pharmacia Biotech Ultraspec 3000 spectropho-tometer (Amersham–Pharmacia Biotech, Boronia, Austra-lia), and the initial enzyme rates were determined. Here 1unit of enzyme activity is equal to the formation of 1 �molof dihydropteroate per minute. For the assay, a series ofreagent mixes where one of the substrate concentrationswas varied (for the determination of Km, Ki, or V) wasmade. BuVers used to make up reagent solutions werepurged with nitrogen to reduce oxidation of the pterins.Final concentrations of reagents in the standard assay wereas follows: 50 mM Tris–HCl (pH 8.5), 1.0 mg/ml bovineserum albumin, 5.0 mM MgCl2, 20 mM �-mercaptoethanol,50 mM NADPH, 50 mM 6-hydroxymethyl-7,8-dihydrop-terin or the pyrophosphate, 50 mM p-ABA, and 1.0 �g/mlDHFR. ATP (Wnal concentration 100 �M) was includedwhen HPPK activity was being measured (with 6-hydroxy-methyl-7,8-dihydropterin as cosubstrate). The sensitivity ofthe assay was measured under these conditions with a seriesof dilutions of the enzyme. Five determinations were car-ried out for each dilution, and the sensitivity was deter-mined as the lowest enzyme concentration where there wasa signiWcantly increased rate of reaction above the uncata-lyzed rate. To determine the intraassay variation, the assaywas repeated a total of 10 times and the means§SD wascalculated. Interassay variation was determined using datafrom 10 standard assays carried out on separate occasions.The activities of DHFR toward both dihydrofolate anddihydropteroate were measured, and the V and Km valueswere determined.

For the 96-well plate assay, Nunc Nunclon plates (cat.no. 167008, Medos, Mount Waverley, Australia) wereused and were read at 340 nm in a Thermo MultiskanAscent plate reader (Pathtech, Box Hill South, Australia)maintained at 37 °C in an incubator. The total volumeused was 150 �l, with 135 �l reagent mixture and 7.5 �ltest compound in water or in DMSO (5% Wnal concentra-tion of DMSO), and the reaction was started by the addi-tion of 7.5 �l HPPK–DHPS (at 20 �g/ml). Plates wereincubated at 37 °C and shaken before monitoring theabsorbance. Each well was read a total of 50 times with8 s between readings (total time 6.67 min). The rate ofdecrease in absorbance at 340 nm for each well was deter-mined after exporting the absorbance data from the platereader software via MS Excel into GraphPad Prism (ver-sion 4.0). To determine the Km values of p-ABA withwild-type enzyme and the four mutant isoform DHPSenzymes, a reagent mixture containing all reactantsexcept p-ABA and the enzymes was used. A series of dilu-tions of p-ABA was made, starting with a 400-mM work-ing dilution and down to a 2.0-�M solution (giving Wnalconcentrations from 20 mM to 0.1 �M in the wells). V andKm values were determined by using Hanes plots [18]. Todetermine binding constants for three sulfa drugs, thewild-type and four mutant alleles of the enzyme were

assayed by varying the concentration of the sulfa drug inthe 96-well plate assay (treating the sulfa drugs as sub-strates). The Km values were determined from the Hanesplots.

To calculate the factor to convert changes in absorptionat 340 nm to enzyme rates in nanomoles per minute, theabsorbance (340 nm) of the products of the reaction(NADP+ and tetrahydropteroate) Wrst must be subtractedfrom the total absorbance of the substrates (NADPH and6-hydroxymethyl-7,8-dihydropterin diphosphate). p-ABAhas no signiWcant absorbance at this wavelength. Theabsorption of a 50-�M solution of the pterin substrate plus50�M NADPH was measured in a 1-cm quartz spectro-photometric cell and in wells of a 96-well plate. This wascompared with the absorption of a solution containing50�M NADP+ and 50 �M tetrahydropteroate. Completeconversion of 50 nmol/ml of substrates to products wouldgive a change in absorption at 340 nm of 0.552 in the UVcell (an eVective molar absorptivity change of 11,040 M¡1

cm¡1) and of 0.298 in the wells. From these values, thechange in absorption was converted to nanomoles per min-ute. The concentrations of the reactants were determinedfrom their molar absorptivity values as follows: pterin sub-strate – �277D20,000 M¡1 cm¡1 [19] and NADPH – �339D6200 M ¡1 cm¡1; NADP+ – �260D 18,000 M¡1 cm¡1; tetrahy-dropteroate (concentration based on that of tetrahydrofo-late) – �297D29,100 M¡1 cm¡1 [20].

Results

The DHPS assay described here is a coupled enzymeassay where the dihydropteroate, formed from the con-densation of 6-hydroxymethyl-7,8-dihydropterin pyro-phosphate with p-ABA catalyzed by DHPS, is reduced totetrahydropteroate by an excess of DHFR with NADPHas the cofactor. These reactions are shown in Fig. 1.DHFR catalyzes the reduction of its natural substratedihydrofolate approximately Wve times faster than it doesdihydropteroate, although the Km values for the two sub-strates are similar (Km D 1.7 �M for dihydrofolate and1.4 �M for dihydropteroate). If HPPK–DHPS is omittedfrom the reaction mixture, there is no reduction of thesubstrate. Similarly, if p-ABA, 6-hydroxymethyl-7,8-dihy-dropterin pyrophosphate, or DHFR is omitted from themixture, there is no reaction. If p-ABA is replaced by asulfa drug—sulfamethoxazole, dapsone, or sulfachloropy-ridazine—there is no observed reaction without DHPS,indicating that the drugs themselves are not reduced byDHFR/NADPH.

The assay can also be used to measure DHPS enzymeactivity using sulfa drugs as substrates in place of p-ABA. If6-hydroxymethyl-7,8-dihydropterin pyrophosphate is incu-bated with a sulfa drug and DHPS, a pterin–sulfa adduct isformed [21]. This can be separated from the starting materi-als by anion exchange chromatography. The adduct is thenreduced by incubation with DHFR and NADPH as fordihydropteroate (results not shown).

230 Rapid assay for dihydropteroate synthase / R.T. Fernley et al. / Anal. Biochem. 360 (2007) 227–234

The limit of detection of the assay as described here is0.05�g/ml of wild-type S. cerevisiae DHPS enzyme (mea-sured over 6 min) and at least 0.2�g/ml for the HPPK activ-ity. The response is linear over at least a 10-fold range ofenzyme concentration for HPPK and a 17.5-fold range forDHPS (Fig. 2). Increasing the HPPK–DHPS concentrationgives a proportional increase in measured activity (r2D0.990for DHPS activity), with a doubling of enzyme concentrationgiving a 1.97-fold increase in activity. For HPPK activity,r2D0.975 and a doubling of enzyme concentration gives a1.98-fold increase in activity. The assay is reproducible, withan intraassay variation of 5.4% (interassay variation 10.6%)for the DHPS activity (nD10) and an intraassay variation of5.7% (interassay variation 11.1%) for HPPK activity (nD10).The V and Km values for DHPS and HPPK activities for thewild-type enzyme are summarized in Table1.

The assay was also used to measure kinetic parametersof four mutant alleles of DHPS. The mutations in thesealleles, constructed in our laboratory, are associated withresistance to sulfa drugs in vivo [16,22]. The mutationsthat aVect the binding of the sulfa drugs to the active siteof DHPS also aVect the binding of p-ABA to this site. TheMichaelis constants (Km) for p-ABA for the four mutantalleles were determined by assaying DHPS activity over awide range of p-ABA concentrations. For example, therates for one allele, ARP DHPS, were measured in a single96-well plate with 28 p-ABA concentrations ranging from0.1 �M to 20 mM. The 11 rates spanning the concentration

range with the greatest eVect on the rates are shownin Fig. 3A. These results are arranged in a Hanes plot(p-ABA concentration against [p-ABA]/enzyme rate) inFig. 3B. The correlation coeYcient for the line of best Wt

Fig. 2. Calibration curve for HPPK and DHPS assays. The HPPK andDHPS activities were assayed separately with increasing concentrationsof the bifunctional enzyme and the enzyme rate (in nmol/min)plotted against the protein concentration. The concentrations of sub-strates (6-hydroxymethyl-7,8-dihydropterin pyrophosphate or 6-hydroxy-methyl-7,8-dihydropterin, p-ABA, and NADPH) were 50 �M, and theconcentration of ATP (HPPK) was 100 �M, in the assay. Initial rates ofthe reaction (maximum of 10% of substrates used) were used to calculatethe enzyme rates. Five replicates were carried out for each enzyme concen-tration. For DHPS activity, r2 D 0.990; for HPPK activity, r2 D 0.975.

0 1 2 3 4

0

1

2

3

DHPS

HPPK

[Enzyme] (mg/ml)

Rat

e (n

mol

/min

)

Fig. 1. Enzyme reactions of the folate biosynthetic pathway involved in the assay. A series of reactions leads to the formation of dihydropteroate that sub-sequently is conjugated to glutamate in a reaction catalyzed by dihydrofolate synthase (DHFS). The resulting dihydrofolate is reduced to tetrahydrofolate(a reaction catalyzed by DHFR). In the assay, the DHFS step is “bypassed,” with the dihydropteroate formed being reduced directly to tetrahydroptero-ate by DHFR/NADPH.

Rapid assay for dihydropteroate synthase / R.T. Fernley et al. / Anal. Biochem. 360 (2007) 227–234 231

was 0.9997. This was repeated for all of the alleles and theKm and V values calculated from the plot, and these arelisted in Table 1.

The assay was used to measure the binding aYnities ofthree sulfa drugs—sulfamethoxazole, sulfachloropyrid-azine, and dapsone—for the wild-type and four mutantalleles. The Km values can be measured directly in the assaywhen the sulfa compounds replace p-ABA as the secondsubstrate. The concentration of 6-hydroxymethyl-7,8-dihy-dropterin pyrophosphate was held constant at 50 �M,whereas the concentrations of the sulfa drugs were varied.Hanes plots were used to calculate the Km and V values,and these are listed in Table 1. The enzyme rate curves forthe three sulfa drugs with wild-type DHPS are shown inFig. 4A, and the corresponding Hanes plot is shown inFig. 4B.

Discussion

The enzymes of the folate biosynthetic pathway havebeen targets for antimicrobials for many decades. Sulfona-mides and sulfones that inhibit DHPS are still commonlyused to treat infectious diseases such as malaria, pneumo-coccal diseases, and urinary tract infections. However, resis-tance to this class of drugs is widespread and increasing [5].

An estimated 2–3 million deaths per year are attributed tomalaria, with another 2 to 3 million deaths being due topneumococcal diseases and still another 2 million being dueto tuberculosis, mostly aVecting young children in thedeveloping world. Thus, there is a growing and pressingneed to develop new and eVective drugs to treat diseasessuch as malaria and tuberculosis. To develop new drugs,one approach is to screen chemical libraries for compoundsthat inhibit or interact with the target enzyme. The pub-lished assays for DHPS and HPPK activities involve incor-poration of radioactively labeled substrates and thin-layerchromatographic separation of the products. This methodis far too slow and laborious to enable a signiWcant numberof compounds to be screened for activity. Recently, amethod based on the use of pyrophosphatase to hydrolyzethe pyrophosphate formed in the DHPS reaction and mea-surement of the inorganic phosphate formed was patented[14]. Although this is a signiWcant improvement on the pre-vious method, it requires a second addition of reagentsafter the incubation step and measures only one time pointper well.

The method described in the current article is a contin-uous microplate assay method that allows rapid andaccurate kinetic analysis of the enzyme reactions. It isbased on coupling the reaction catalyzed by DHPS to the

Table 1Kinetic parameters for yeast HPPK–DHPS (wild-type and mutant alleles) as determined by the coupled spectrophotometric method

Note. SMX, sulfamethoxazole; SCP, sulfachloropyridazine; wt, wild-type.a 6-Hydroxymethyl-7,8-dihydropterin pyrophosphate as substrate.b 6-Hydroxymethyl-7,8-dihydropterin as substrate.

Enzyme V (U.mg¡1) Km (p-ABA) (�M) Km (pterins) (�M) Ki (SMX) (�M) Ki (SCP) (�M) Ki (dapsone) (�M)

wt DHPS 1.32 § 0.11 3.4§ 0.3 2.4 § 0.3a 8.5§ 1.0 2.95 § 0.15 0.68 § 0.07wt HPPK 0.27 § 0.03 – 0.39§ 0.03b – – –TRS DHPS 0.61 § 0.06 48.5 § 3.9 – 54.3 § 3.8 51.3 § 6.1 14.8 § 0.7ARP DHPS 0.33 § 0.02 103§ 8.0 – 3.71 § 0.33 6.3 § 0.5 2.05 § 0.16VRS DHPS 0.14 § 0.02 1611§ 112 – 760§ 75 940 § 56 1,290 § 90ARS DHPS 0.083§ 0.01 4020§ 301 – 1170 § 59 960 § 86 1,160 § 69

Fig. 3. Determination of the Km for p-ABA for the yeast bifunctional HPPK–DHPS enzyme with a single mutation at T557 (ARP DHPS). A range of con-centrations of p-ABA, from 0.1 �M to 20 mM, was assayed in triplicate for DHPS activity in a single 96-well plate. DHPS substrates 6-hydroxymethyl-7,8-dihydropterin pyrophosphate and NADPH were 50 �M. The concentration of the DHPS was 6.1 �g/ml in the wells. The plate was incubated at 37 °C, andthe A340 was read over 6 min. The individual rates for 10 �M to 2.5 mM p-ABA, where the greatest change in enzyme rate with change in p-ABA concen-tration occurred, are shown in (A). The Hanes plot (p-ABA concentration against [p-ABA]/enzyme rate) is shown in (B). The Km for p-ABA for this alleleof DHPS, calculated from the Hanes plot, was 103 �M.

0 500 1000 1500 2000 25000

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232 Rapid assay for dihydropteroate synthase / R.T. Fernley et al. / Anal. Biochem. 360 (2007) 227–234

oxidation–reduction of NAD(P)H–NAD(P)+, which iscommonly used in assays and is well understood [23,24].The assay depends on the ability of DHFR (whose usualsubstrate is dihydrofolate) to reduce dihydropteroate totetrahydropteroate. That this reaction takes place hasbeen demonstrated previously using E. coli DHFR [2],where the product of the reaction was identiWed bynuclear magnetic resonance (NMR) spectroscopy.Although dihydropteroate is not as good a substrate asdihydrofolate, excess DHFR added to the reactionensures that this is not the rate-limiting step of the cou-pled reaction. The assay has been applied to measure theactivities of HPPK and DHPS, and theoretically it couldbe used to measure the activity of DHNA, which catalyzesthe reaction preceding that catalyzed by HPPK in thefolate pathway. The assay as described here has been setup to measure initial enzyme rates to give accurate mea-surements of kinetic properties of DHPS enzymes and ofinhibitor constants. Faster reading of the 96-well platescould be used for screening for enzyme inhibitors if highaccuracy of enzyme rates is not required.

The parameters of DHPS enzyme activity reported hereare similar to those obtained with the radioactivity assay(incorporation of radioactively labeled p-ABA into thepterin substrate) [10]. Enzyme activity (V) of DHPS fromdiVerent species ranges from 0.011 U/mg for the P. falcipa-rum DHPS with a Km for p-ABA of 1.25 �M [13] to0.6 U/mg for the Mycobacterium tuberculosis enzyme with aKm for p-ABA of 0.37�M [25] and 1.32 U/mg (KmD 3.4�Mfor p-ABA) reported here. Resistance to sulfa drugs in, forexample, Pneumocystis jirovecii is associated with muta-tions in the DHPS gene at T517 (to Ala [equivalent to T557 inthe S. cerevisiae trifunctional protein]) and P519 (to Ser[P559]) [26]. The presence of both mutations (A517RS519) in aclinical isolate is associated with a higher degree of resis-tance. A laboratory isolate with a double mutation,V517RS519, also has greatly increased resistance [16]. Thisincreased resistance to sulfa drugs is reXected in the bindingconstants of p-ABA to the enzyme’s active site. The singlemutant alleles have p-ABA binding constants 14- to 30-foldhigher than the value for the wild-type enzyme, whereas the

double mutant alleles have Km values 500- to more than1000-fold higher than the value for the wild-type enzyme.

The binding constants of three sulfa drugs in commonuse were measured with wild-type and four mutant alleles.The method used to measure the Km values was the kineticassay with the pterin pyrophosphate and sulfa drugs assubstrates but without p-ABA. The Km values determinedhere are equivalent to the Ki values where the inhibitor ofthe enzyme is also a substrate for the enzyme [18]. Otherresearchers have measured Ki values by varying bothp-ABA and sulfa drug concentrations [13,27]. However,the sulfa drugs take part in the DHPS reaction and areconverted to pterin adducts [21], making interpretation ofthe results from this method diYcult, especially if thereaction rates for p-ABA and sulfa compounds are similar(as they are shown to be here). The kinetic constants weremeasured in this study by treating the sulfa drugs as thesubstrate and using Hanes plots to measure the Km values[18,28].

For the single mutant allele, TRS DHPS, the Km valuesfor the sulfa drugs are 6- to 22-fold higher than the valuefor the wild-type enzyme. For the double mutant alleles,the values are 89 to 1900 times the value for the wild-typeenzyme. For the second single mutant allele (ARPDHPS), the results were anomalous, with the enzymebeing from 2.3-fold more sensitive (sulfamethoxazole) to3-fold less sensitive (dapsone), as measured by the Km val-ues. This is in agreement with our earlier data showingthat for ARP DHPS (the S. cerevisiae DHNA–HPPK–DHPS expressed in E. coli), the minimum inhibitory con-centration of drug required to give 100% inhibition ofgrowth was 4-fold lower for sulfamethoxazole than forwild-type DHPS (whereas the other mutant allelesrequired higher concentrations of drugs for this inhibi-tion) [16]. The results for sulfamethoxazole and sulfachlo-ropyridazine show that the two compounds have similaraYnities for the wild-type and mutant isoforms of theenzyme. Previous studies on whole cells have shown thatthe cells are more sensitive to sulfachloropyridazine inhi-bition, and this may be the result of diVerent permeabili-ties of the cells to the two drugs [26].

Fig. 4. Determination of the binding constants for the three sulfa drugs—dapsone, sulfachloropyridazine (SCP), and sulfamethoxazole (SMX)—to yeastwild-type DHPS. The enzyme was incubated with diVerent concentrations of the sulfa drugs in the 96-well assay. The measured rates are plotted againstthe substrate concentrations (A) or the substrate concentration/rate against substrate concentration (Hanes plot) (B). The binding constants for the threesulfa drugs were read from the plot (where the intercept on the x-axis D –Km).

0 100 200 300 400 500

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v (U

/mg)

BA

Rapid assay for dihydropteroate synthase / R.T. Fernley et al. / Anal. Biochem. 360 (2007) 227–234 233

In most cases, the mutations that lead to decreased bind-ing of p-ABA to the enzyme also result in decreased aYnityof the sulfa drugs, with the exception being the ARS muta-tion. Although p-ABA and the sulfa drugs bind to the samepocket in the DHPS active site, there are diVerences in theorientation of the binding of these substrates and diVer-ences in the side chains in the active site interacting withthese substrates [29].

This DHPS assay was found to be robust and straight-forward with attributes of high sensitivity, low variation,rapid assays, and rapid data analysis. The assay was used ina 96-well format to measure kinetic properties of DHPSand inhibition of the enzyme activity by sulfa drugs. Theassay could be readily miniaturized further to a 384-wellformat and, with robotic delivery of reagents, would besuitable for rapid screening of chemical libraries to Wndinhibitors of the enzymes activities. Because DHPS andDHFR catalyze reactions involving closely related mole-cules, potential inhibitors of DHPS might also inhibitDHFR. To determine which activity (if not both activities)is being inhibited in this assay, a separate assay withDHFR, dihydrofolate substrate, and potential inhibitorwould be included in any screen for inhibitors. Thus, thisassay (not patented) oVers a methodology to aid researchand development into replacement drugs for diseases suchas malaria, pneumococcal diseases, PCP, and perhapstuberculosis that are ravaging populations in developingnations.

Acknowledgments

We thank Tom Peat and Lance Macaulay for criticalreviews of the manuscript. This work was supported in partby the National Institutes of Health through the Universityof North Carolina at Chapel Hill (Grant 1 RO1AI46966-01A1 to Steven Meshnick).

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