Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway

It has been proposed that isoprenoid biosynthesis in severalgram-positive cocci depends on the mevalonate pathway for conversionof acetyl coenzyme A to isopentenyl diphosphate. Mevalonatekinase catalyzes a key reaction in this pathway. In this studythe enzyme from Staphylococcus aureus was expressed in Escherichiacoli, isolated in a highly purified form, and characterized.The overall amino acid sequence of this enzyme was very heterologouscompared with the sequences of eukaryotic mevalonate kinases.Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand analytical gel filtration chromatography suggested thatthe native enzyme is a monomer with a molecular mass of approximately33 kDa. The specific activity was 12 U/mg, and the pH optimumwas 7.0 to 8.5. The apparent K_m values for R,S-mevalonate andATP were 41 and 339 µM, respectively. There was substantialsubstrate inhibition at millimolar levels of mevalonate. Thesensitivity to feedback inhibition by farnesyl diphosphate andits sulfur-containing analog, farnesyl thiodiphosphate, wascharacterized. These compounds were competitive inhibitors withrespect to ATP; the K_i values were 46 and 45 µM for farnesyldiphosphate and its thio analog, respectively. Parallel measurementswith heterologous eukaryotic mevalonate kinases indicated thatS. aureus mevalonate kinase is much less sensitive to feedbackinhibition (K_i difference, 3 orders of magnitude) than the humanenzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP,a fluorescent substrate analog, suggesting that there are similaritiesin structural features that are important for catalytic function.

INTRODUCTION

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Introduction

During isoprenoid biosynthesis in most eubacteria the methylerythritol 4-phosphate pathway is used for production of isopentenyldiphosphate. In contrast, animals, yeast, and archaea produceisopentenyl diphosphate by the better-characterized mevalonatepathway. Recently, it has been reported (23) that the genomesof several gram-positive cocci encode enzymes of the mevalonatepathway and that survival of these bacteria requires this pathway.The genes that have been proposed to encode enzymes of the mevalonatepathway in gram-positive cocci are heterologous with the codingsequences for comparable enzymes in higher organisms. However,the function of the mevalonate pathway in these bacteria issupported by characterization of Enterococcus faecalis 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) synthase (20), as well as a dual-function protein,acetoacetyl-CoA thiolase/HMG-CoA reductase (9). Recently, phosphomevalonatekinase activity has been observed for a protein from Streptococcuspneumoniae (15). In contrast, there has not been substantialcharacterization of mevalonate kinase isolated from any gram-positivecoccus.

Mevalonate kinase (EC 2.7.1.36) catalyzes the following reaction(the reaction is divalent cation dependent, and Mn²⁺ supports25% of the activity measured with Mg²⁺):

This enzyme is a key enzyme (22) in the mevalonate pathway forbiosynthesis of isopentenyl diphosphate. In fact, genetic defectsthat decrease enzyme activity in humans (11) correlate withinherited diseases such as mevalonic aciduria and hyperimmunoglobulinD syndrome. Such observations underscore the ability of mevalonatekinase to influence isoprenoid biosynthesis in animals. Theenzyme in gram-positive bacteria has not been as extensivelycharacterized as its counterpart in animals and plants (2, 16,17, 19, 21), and given the significant differences between thepredicted amino acid sequences of animal and bacterial proteins,significant differences between the properties of theses enzymesseem probable. Such differences could make bacterial mevalonatekinase an attractive target for design of antibiotics.

In this report we describe expression, isolation, and characterizationof Staphylococcus aureus mevalonate kinase. Our observationsprovide support for the function of the mevalonate pathway ingram-positive bacteria. A comparison of the properties of thebacterial enzyme with the properties of animal mevalonate kinasesprovided insight into some differences between isoprenoid metabolismin prokaryotes and isoprenoid metabolism in eukaryotes. Thecontrasts in feedback inhibition between the heterologous enzymesare examples of such differences.

MATERIALS AND METHODS

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Materials and Methods

Materials. Escherichia coli BL21(DE3) cells were obtained from Novagen.DNA purification kits were purchased from Qiagen and Sigma.A pET-23a plasmid, modified to include the open reading frameproposed to encode S. aureus mevalonate kinase as an insert(untagged and unfused) between the NdeI and EcoRI restrictionsites, was a generous gift from Imogen Wilding and Mehul Patel(GlaxoSmithKline). Recombinant human and rat mevalonate kinaseswere expressed and isolated as previously described (16, 17).Low-molecular-weight protein sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) markers were obtained from Bio-Rad.2'(3')-O-(2,4,6-Trinitrophenyl)ATP (TNP-ATP) was obtained fromMolecular Probes. Farnesyl diphosphate (FPP) was purchased fromEchelon Biosciences (Salt Lake City, Utah). Farnesyl thiodiphosphate(FSPP) was a generous gift from C. D. Poulter (University ofUtah). ATP, ß-NADH, dithiothreitol (DTT), phosphoenolpyruvate,DL-mevalonic acid lactone, and other reagents were purchasedfrom Sigma, unless specified otherwise.

Mevalonate kinase assay. Routine measurement of enzyme activity was performed spectrophotometricallyat 30°C by using a 1.0-ml mixture which contained 100 µmolof HEPES (pH 7.5), 100 µmol of KCl, 0.2 µmol ofphosphoenolpyruvate, 0.5 µmol of DTT, 0.16 µmolof NADH, 10 µmol of MgCl₂, 4 U of lactate dehydrogenase,4 U of pyruvate kinase, 5.0 µmol of ATP, and 0.8 µmolof DL-mevalonate. Activity was calculated by using the extinctioncoefficient for NADH at 340 nm (6.22 cm^-1 mM^-1). Specific activitywas expressed in units of enzyme activity per milligram of protein,where 1 U corresponded to formation of 1 µmol of productmin^-1. The protein concentration was determined by the Bradfordassay (4) by using bovine serum albumin as the standard.

Expression and purification of S. aureus mevalonate kinase. A single colony of E. coli BL21(DE) containing the S. aureusmevalonate kinase expression plasmid pSaMK, obtained from aplate containing Luria-Bertani medium with ampicillin (0.1 mg/ml),was used to inoculate 10 ml of the same medium, which was incubatedovernight at 37°C. The resulting culture was used to inoculate1 liter of Luria-Bertani medium containing ampicillin that wasincubated at 22°C for 24 h with shaking at 200 rpm. Thenthe cells (A₆₀₀, 3.8) were induced with 1 mM IPTG (isopropylthiogalactoside)and harvested after 7 h of induction (A₆₀₀, 4.7 to 5.2) by centrifugation(4,000 x g; 15 min). The cells were suspended in a buffer containing20 mM potassium phosphate buffer (pH 7.5) 0.5 mM DTT, 0.5 mMphenylmethylsulfonyl fluoride, and 1 µg of DNase per mland were lysed by passage twice through a French pressure cellat 11,000 lb/in². The lysate was centrifuged at 100,000 x gfor 1 h, and the supernatant was dialyzed overnight at 4°Cagainst 4 liters of buffer containing 20 mM potassium phosphatebuffer (pH 7.5) and 0.5 mM DTT.

The dialysate was applied to a Sephadex Fast Q anion-exchangecolumn (1.5 by 35 cm) equilibrated with the buffer used fordialysis. The column was washed until the A₂₈₀ of the effluentwas <0.2. Proteins were eluted by using a 1-liter gradientof 20 to 100 mM potassium phosphate buffer (pH 7.5) with 0.5mM DTT. Mevalonate kinase eluted in a highly purified form (Fig.1) as a sharp peak early in the gradient. Fractions containingpeak enzyme activity that were electrophoretically pure werepooled and concentrated by ultrafiltration to a concentrationof approximately 2 mg/ml. Approximately 75% of the activityin the dialyzed supernatant was recovered in the pooled anion-exchangechromatography fractions. Fivefold purification was accomplishedby anion-exchange chromatography, and on average, the yieldwas 90 mg of enzyme with a specific activity of 12.4 ±0.9 U/mg (mean ± estimated error) at 30°C.

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FIG. 1. SDS-PAGE of S. aureus mevalonate kinase at different stages of purification. A 12.5% polyacrylamide gel was electrophoresed under denaturing conditions, and protein was detected by staining with Coomassie brilliant blue. Lanes 2 and 3 contained 12 µg of protein, and lane 4 contained 5 µg of protein. Lane 1, molecular mass standards, including phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31kDa), trypsin inhibitor (21.5kDa), and lysozyme (14.4 kDa); lane 2, soluble fraction after bacterial disruption with a French pressure cell; lane 3, dialyzed soluble bacterial extract; lane 4, eluate from a Sephadex Fast Q column.

Kinetic studies. To determine the maximal velocities (V_max) and Michaelis constants(K_m) for substrates, the ATP concentration was varied from 0.02to 5 mM and the mevalonic acid concentration was varied from0.02 to 0.60 mM. Higher mevalonic acid concentrations were employedwhen substrate inhibition by this metabolite was investigated.To determine steady-state kinetic constants, data were subjectedto nonlinear regression fits to the Michaelis-Menten equation(13) by using the Grafit programs (Erithacus Software). To determinethe inhibition constants for FPP and FSPP, the same programwas used for linear fits of slopes (from double-reciprocal plots)as a function of inhibitor concentration.

RESULTS

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Results

Native and subunit molecular weights of S. aureus mevalonate kinase. The SDS-PAGE mobility of denatured S. aureus mevalonate kinase,when plotted on a calibration curve constructed for molecularmass standards, suggested that the subunit molecular mass was33.1 kDa under the denaturing conditions used. This estimateis in good agreement with the molecular mass (32.9 kDa) calculatedfrom the amino acid composition deduced from the open readingframe. Analytical gel filtration was performed under nondenaturingconditions by using a Superose 12 fast protein liquid chromatography(FPLC) column equilibrated with 50 mM HEPES (pH 7.50 containing0.15 M KCl). Based on a calibration curve for molecular massstandards (Fig. 2), the elution properties of the enzyme, whichproduced a single symmetric peak, suggested that the molecularmass is 34.5 kDa, which is consistent with the molecular massof a monomeric native enzyme.

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FIG. 2. Analytical gel filtration chromatography of S. aureus mevalonate kinase. The line is the calibration curve used to estimate the native molecular weight based on the elution position during analytical gel filtration from a Superose 12 FPLC column (1 by 30 cm). Analytical gel filtration was performed by using a Superose 12 10/30 FPLC column (Pharmacia) equilibrated with 50 mM HEPES (pH 7.5) containing 0.15 M KCl. The flow rate was 0.4 ml/min, and 100 µl of protein (1 to 3 mg/ml) was applied; protein elution was monitored by determining the absorbance at 280 nm. The void volume was determined by using blue dextran. The calibration curve is a plot of log molecular weight (log Mw) of protein versus ratio of the elution volume to the void volume (Ve/Vo). The molecular weight standards that were used to estimate the native molecular weight included bovine serum albumin (molecular weight, 66,000) ( ${diamond}$ ), ovalbumin (45,000) ( ${circ}$ ), carbonic anhydrase (31,000) ( ${triangledown}$ ), and chymotrypsinogen (21,000) ( ${square}$ ). •, mevalonate kinase.

Kinetic characterization of S. aureus mevalonate kinase. Steady-state rate estimates that were obtained by using a rangeof ATP concentrations and a fixed, noninhibitory level of R,S-mevalonicacid (0.44 mM) indicated that the apparent K_m was 339 ±23 µM (mean ± estimated error) for ATP. Replottingof data sets collected at several different fixed levels ofmevalonic acid indicated that the limiting or true K_m was 136µMfor ATP. Similarly, measurements of the rate that were obtainedby using a fixed ATP concentration (5 mM) and R,S-mevalonatelevels that ranged from 0.02 to 0.60 mM indicated that the apparentK_m was 41 ± 3 µM (mean ± estimated error)for mevalonate. Analysis of data sets collected at differentfixed levels of ATP indicated that the limiting or true K_m was35 µM for mevalonate. At higher levels of mevalonate,substrate inhibition was observed. A double-reciprocal plotof rate versus mevalonate concentration (including the rangeat which substrate inhibition was evident) amply demonstratedthe inhibitory effect (Fig. 3). The concentration required fora 50% decrease compared with the optimal rate was estimatedto be 2.88 ± 0.37 mM (mean ± estimated error).A test performed with mevalonate 5-diphosphate indicated thatthere was no inhibition at concentrations up to 0.8 mM.

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FIG. 3. Substrate inhibition of S. aureus mevalonate kinase. A plot of 1/rate (1/ ${nu}$ ) versus 1/[R,S-mevalonate]^-1 (1/[R,S-MVA]) is shown. The experiments were performed with 4.5 mM ATP, 10 mM Mg²⁺, and different concentrations of R,S-mevalonate (0.022 to 2.18 mM). A 50% decrease in the optimal rate was calculated to correspond to a mevalonate concentration of 2.88 mM.

Product inhibition experiments in which mevalonate 5-phosphatewas tested against ATP at a fixed, unsaturating level of mevalonicacid (92 µM) indicated that there was mixed, noncompetitiveinhibition (K_i(slope) = 1.98 mM; K_i(intercept) = 4.43 mM). Mixed,noncompetitive inhibition was also observed when mevalonate5-phosphate was tested with mevalonate at a fixed unsaturatinglevel of ATP (0.139 mM) (K_i(slope) = 2.23 mM; K_i(intercept)= 2.87 mM). In contrast, an uncompetitive inhibition patternfor mevalonate 5-phosphate was observed when a fixed, saturatinglevel of ATP (7.5 mM) was employed (K_i = 1.56 mM). These productinhibition patterns are diagnostic for an ordered sequentialmechanism in which the substrate mevalonate binds before ATPand the product mevalonate 5-phosphate is released before ADP.In this respect, the S. aureus enzyme has a kinetic mechanismsimilar to that reported for the hog liver enzyme (2).

A plot of the pH dependence of V_max, extrapolated from dataobtained with different concentrations of ATP (Fig. 4), indicatedthat there was a broad optimum pH range (pH 7.0 to 8.5). A similarpH profile was obtained when V_max was estimated by extrapolationfrom data obtained with different concentrations of mevalonate.

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FIG. 4. pH optimum for S. aureus mevalonate kinase. V_max was determined by the spectrophotometic assay with variable ATP concentrations. The reaction was studied over a pH range from 5.5 to 9.5 by utilizing morpholineethanesulfonic acid (MES), HEPES, N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS), and 3-N-cyclohexylamino-2-hydroxypropane sulfonic acid (CAPSO) buffers in an overlapping manner. At the extremes of the pH range studied, additional coupling enzymes were utilized to ensure that accurate estimates of mevalonate kinase activity were obtained. Mevalonate kinase stability was evaluated over the pH range to ensure that no significant loss of activity occurred over the time course of the kinetic measurements. Assay mixtures containing all the components except the variable substrate, coupling enzymes, and mevalonate kinase were prepared, and the pH value was adjusted and confirmed to be the reported value; the pH value was reconfirmed after the remaining reagents were added and the initial rate was determined. The steady-state parameters at each pH were determined by nonlinear regression fits of the data. Calculated fits of V_max as a function of pH were determined by using the program TableCurve 2D to fit the data to the following equation: where y is V_max, C_o is the pH-independent plateau value, [H⁺] is the proton concentration, K₁ is the ionization constant for an acidic group, and K₂ is the ionization constant for a basic group (6). The theoretical curve shown as the best fit to the data was calculated by using pK values of 6.1 and 9.4 for ascending and descending limbs, respectively. Due to the limited pH range over which the enzyme is stable, these pK values (which fall either close to the limit or outside the pH range over which data could be collected) should not be interpreted as suggesting pK values for ionization of amino acid side chains or substrate functional groups but rather reflect the values that produced the best curve fit to data. The broad pH optimum for enzyme activity is pH 7.0 to 8.5.

The rate of the mevalonate kinase reaction was measured as afunction of the ATP concentration by using several fixed concentrationsof FPP or FSPP (14). In both cases, competitive inhibition withrespect to ATP was observed (Fig. 5). Secondary plots of slopeversus inhibitor concentration indicated that the K_i valueswere 46 µM for FPP and 45 µM for FSPP (Fig. 6).For comparative purposes, Fig. 6 also shows similar slope-versus-inhibitorplots for data generated by using human and rat mevalonate kinases.The results (Table 1) underscore the large difference in sensitivityto feedback inhibition between heterologous mevalonate kinasesfrom prokaryotic and eukaryotic sources. Preliminary measurementsfor purified E. faecalis mevalonate kinase (provided by M. Hedland V. W. Rodwell, Purdue University) suggested that this enzymefrom another gram-positive bacterium exhibits sensitivity toFPP inhibition (K_i = 45 µM) that is comparable to thatof the S. aureus enzyme.

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FIG. 5. Inhibition of S. aureus mevalonate kinase by FPP (top panel) and FSPP (bottom panel). The rate of mevalonate kinase activity was measured as a function of ATP concentration without inhibitors, as well as with several fixed concentrations of FPP or FSPP. Double-reciprocal plots of enzyme activities (measured by using 4.2 µg of purified enzyme) as a function of ATP concentration are shown. The ATP concentration ranged from 0.1 to 1.25 mM for the FPP experiments and from 0.2 to 1.25 mM for the FSPP experiments. The FPP concentrations used were 0 µM ( ${circ}$ ), 15 µM ( ${square}$ ), 30 µM ( ${blacksquare}$ ), 45 µM ( ${triangleup}$ ), and 60 µM ( ${blacktriangleup}$ ). The FSPP concentrations used were 0 µM ( ${circ}$ ), 24.5 µM (•), 49 µM ( ${diamond}$ ), and 73.5 µM ( ${diamondsuit}$ ).

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FIG. 6. Comparison of FPP inhibition and FSPP inhibition of recombinant S. aureus (A), human (B), and rat (C) mevalonate kinases. The rate of mevalonate kinase activity was measured as a function of the ATP concentration without FPP and with several fixed concentrations of either FPP ( ${circ}$ ) or FSPP (•). Secondary plots show the slopes from double-reciprocal plots versus corresponding inhibitor concentrations. The ATP concentrations used ranged from 0.1 to 1.25 mM for FPP inhibition and from 0.2 to 1.25 mM for FSPP inhibition. The concentrations of FPP used were 15, 30, 45, and 60 µM for S. aureus mevalonate kinase; 50, 70, 100, and 200 nM for human mevalonate kinase; and 100, 400, and 1,000 nM for rat mevalonate kinase. The concentrations of FSPP used were 24.5, 49, and 73.5 µM for the S. aureus enzyme; 40, 75, and 100 nM for the human enzyme; and 400, 700, and 1,000 nM for the rat enzyme. The estimated K_i values of FPP for the S. aureus, human, and rat enzymes are 46 µM, 35 nM, and 348 nM, respectively. The estimated K_i values of FSPP for the S. aureus, human, and rat enzymes are 45 µM, 29 nM, and 473 nM, respectively.

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TABLE 1. Feedback inhibition of mevalonate kinase

Binding of a fluorescent ATP analog. Previous studies (5) demonstrated that TNP-ATP, a fluorescentanalog of the substrate ATP, can be used as a tight bindingagent for titration of human mevalonate kinase. A test of thebinding of this reagent to S. aureus mevalonate kinase indicatedthat this protein also is titrated by the reagent (Fig. 7).Binding correlated with a strong blue shift of the emissionmaximum (from 557 to 535 nm) for this fluorescent analog, suggestingthat there is a nonpolar environment for TNP-ATP in its binarycomplex with the enzyme. Extrapolation of lines fitted to thelow- and high-occupancy regions of the titration data indicatedthat the binding stoichiometry was 0.62 TNP-ATP molecule perenzyme subunit, a value which is comparable to the estimatefor the heterologous human mevalonate kinase. Thus, bindingdeterminants for TNP-ATP may be similar despite the lack ofstrong overall sequence homology.

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FIG. 7. Fluorescence titration of S. aureus mevalonate kinase with TNP-ATP. An Aminco SLM 4800C spectrofluorimeter was used to obtain measurements; the excitation wavelength was 408 nm, and the emission spectra were scanned at wavelengths from 500 to 600 nm. Tris-HCl buffer (10 mM; pH 7.5) was used in all experiments. For data analysis, the values measured at a fluorescence emission peak of 535 nm for enzyme-bound TNP-ATP were corrected for free TNP-ATP fluorescence; thus, the enhancement of fluorescence is shown, and these data were used in the binding analyses. Sequential additions of TNP-ATP were made to a fluorescence cuvette containing the enzyme site at a concentration of 3 µM. The binding stoichiometry for nonequilibrium complexes was determined (1, 7, 18) from the intersection point of lines fitted to the low-occupancy and plateau regions of the titration data by linear regression analyses. The calculated binding stoichiometry for TNP-ATP (0.62) reflects the number of binding sites per 33-kDa subunit. MK, mevalonate kinase.

DISCUSSION

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Discussion

The deduced sequences of the proposed prokaryotic mevalonatekinases exhibit interesting similarities and differences withthe sequences of eukaryotic forms of the enzyme. For example,key amino acids that influence substrate binding or catalyticefficiency (Fig. 8) include invariant lysine (16), aspartate(17), glutamate (17), and serine (5) residues. Structural results(8, 24) have confirmed that these residues are situated at theactive site. In the case of S. aureus mevalonate kinase, theseresidues are lysine-12, aspartate-145, glutamate-134, and serine-102(Fig. 8). The importance of aspartate-145 is supported by theproperties of the D145C mutant, which substitutes a thiolateanion for the carboxylate; a 65-fold decrease in specific activity(Rios and Voynova, unpublished observations) confirmed the importanceof the active site carboxylate. The S. aureus enzyme, like manyother putative prokaryotic mevalonate kinases, is shorter (306residues, 32.9 kDa) than the eukaryotic enzymes, which are proteinsthat contain approximately 400 residues. Despite the shorterprotein sequence, our results with the isolated enzyme demonstratethat S. aureus mevalonate kinase is very effective in catalyzingphosphorylation of mevalonic acid (see above). In this context,it is interesting that in prokaryotic mevalonate kinases thereis not a C-terminal region corresponding to the region whichharbors human mevalonate kinase valine-377; mutation of thisresidue has been suggested to account for inherited disease(10). S. aureus mevalonate kinase contains two cysteines (residues196 and 234). These amino acids are accessible to dithiobisnitrobenzoicacid modification, which virtually eliminates enzyme activity.However, a comparison of animal, plant, fungal, and bacterialmevalonate kinases indicated that there are no invariant cysteines,suggesting that these residues are not crucial for catalyticfunction even though modifications of them impair function.In this context, mutagenic replacement of these residues (C196S,C234S/A/N) indicated that C196 is not crucial for enzyme functionbut suggested that replacement of C234 by serine, alanine, orasparagine results in an unstable protein (Rios, unpublishedobservations).

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FIG. 8. Conservation of catalytic residues in S. aureus and eukaryotic mevalonate kinases.

In addition to the differences between the subunit sizes ofprokaryotic mevalonate kinases and the subunit sizes of eukaryoticmevalonate kinases, there may also be differences in quaternarystructure. The eukaryotic mevalonate kinases (2, 16, 17, 19,21) appear to be dimers under native conditions, and a dimericrat protein has been observed under crystalline conditions (8).The situation is less clear for the prokaryotic proteins. TheHis-tagged form of the Methanococcus jannaschii enzyme has beenproposed to be a dimer (12). However, in the crystalline state(24), a monomer has been observed. Based on our gel filtrationdata, the untagged, unfused S. aureus mevalonate kinase appearsto be a monomeric species under native conditions. It remainsto be established whether a consensus will develop concerningthe quaternary structure of the prokaryotic enzymes. Certainly,based on functional studies (5, 16, 17) and the crystal structureof animal mevalonate kinase (8), there appears to be no reasonwhy a monomer could not be functional.

There is considerable variability in the reported efficacy offeedback inhibition of mevalonate kinase by FPP. This may bedue to the use of different assay methods, assay buffers, orsources of inhibitor in studies. Both the rat (21) and M. jannaschii(12) enzymes have been reported to be sensitive to inhibitionby micromolar levels of FPP. A report for human mevalonate kinase(17) suggested that there was effective inhibition at much lowerlevels (10^-8 M). In this study, parallel measurements of S.aureus, human, and rat enzymes indicated that there was a genuinedifference in sensitivity to inhibition not only by FPP butalso by the analog FSPP, which inhibits as well as the authenticmetabolite (Table 1). There is an approximate difference of3 orders of magnitude between the K_i values for the prokaryoticenzyme (46 µM for FPP, 45 µM for FSPP) and the K_ivalues for the human enzyme (35 nM for FPP, 29 nM for FSPP).While the rat enzyme is characterized by slightly elevated K_ivalues compared with the values for human mevalonate kinase,there is a difference of approximately 2 orders of magnitudebetween the K_i values for the prokaryotic and rat enzymes. WhileFPP is a competitive inhibitor with respect to ATP for bothprokaryotic and eukaryotic enzymes, the molecular basis forits effect remains to be established. It may be anticipatedthat the inhibitor competes for the ATP site because the phosphorylgroups bind in the region normally occupied by the alpha, beta,and/or gamma phosphoryls of ATP. However, the high affinityexhibited upon FPP binding to animal mevalonate kinase suggeststhat the farnesyl moiety contributes to the binding energy andraises a question concerning its binding interactions. Superpositionof the molecular structures of rat mevalonate kinase (8), whichbinds FPP reasonably tightly, and the M. jannaschii enzyme (24),which binds this inhibitor much more weakly (12), revealed someoverall differences between these proteins, but much smallerdifferences in the area around the ATP site were detected. Thus,structural differences between the prokaryotic and eukaryoticproteins do not immediately suggest a binding locus for thefarnesyl moiety. The utility of comparisons of these structuresis limited since there is no structure available for mevalonatekinase with both ATP and phosphoryl acceptor sites occupied.Additionally, comparisons that are relevant to the S. aureusenzyme are complicated by the fact that this enzyme does notexhibit high overall homology to either the animal or the M.jannaschii enzyme. More information concerning the orientationof FPP bound to either the prokaryotic enzyme or the eukaryoticenzyme or both would be useful for addressing the differencesin efficacy of the inhibitor. Nonetheless, the feedback inhibitionresults underscore the functional differences between the highlyheterologous prokaryotic and eukaryotic mevalonate kinases.In recent years, work on HMG-CoA reductase proteins has ledto the conclusion that there are two classes of enzymes (3);these classes correlate with different sensitivities to thestatin class of HMG-CoA inhibitors. Perhaps additional studyof prokaryotic and eukaryotic mevalonate kinases will resultin similar assignments of these enzymes to classes that correlatewith functional differences. Given the development of S. aureusstrains that are resistant to antibiotics that previously wereefficacious, it seems possible that functional differences betweenhuman and S. aureus mevalonate kinases might be exploited inan attempt to remedy resistance to existing antibiotics.

ACKNOWLEDGMENTS

We are grateful to Imogen Wilding and Mehul Patel for providingthe expression plasmid used for production of recombinant S.aureus mevalonate kinase. We also thank C. Dale Poulter forthe gift of FSPP.

This work was supported in part by NIH grant DK 53766

FOOTNOTES

* Corresponding author. Mailing address: Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Phone: (414) 456-8437. Fax: (414) 456-6570. E-mail: miziorko{at}mcw.edu.

Present address: GlycoFi Inc., Lebanon, NH 03766.

REFERENCES

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