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Published online before print March 31, 2005, 10.1110/ps.041210405
Protein Science (2005), 14:1134-1139. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Enterococcus faecalis phosphomevalonate kinase

Stephanie S. Doun1, John W. Burgner, II2, Scott D. Briggs1,3,4 and Victor W. Rodwell1

1 Department of Biochemistry, 2 Department of Biological Sciences, and 3 Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA4 Walther Cancer Institute, Indianapolis, Indiana 46202, USA

Reprint requests to: Victor W. Rodwell, Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907-2063; e-mail: vrodwell{at}purdue.edu; fax: (765) 494-7897.

(RECEIVED November 2, 2004; FINAL REVISION February 15, 2005; ACCEPTED February 15, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2 [EC] ). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni++ affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37°C. The activation energy was ~5.6 kcal/mol. Activity with Mn++, the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). Km values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 µmol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

Keywords: phosphomevalonate kinase; isoprenoid biosynthesis; mevalonate pathway; 5-phosphomevalonate; mevalonate 5-phosphate; mevalonate 5-diphosphate; isopentenyl diphosphate; Enterococcus faecalis

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041210405.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
The emergence of vancomycin-resistant strains of Staphylococcus aureus (Walsh and Howe 2002) and Enterococcus faecalis (Paulsen et al. 2003), organisms responsible for infective endocarditis, other nosocomial infections, and food poisoning, has exhausted the armamentarium of antibiotics suitable for control of infections by these Gram-positive cocci. As noted by Ziglam and Nathwani (2003), the emergence worldwide of vancomycin-resistant entero-cocci is a grave concern, and new agents are essential to meet this threat. Inhibiting the biosynthesis of isopentenyl diphosphate (IPP), the building block of all isoprenoids, offers potential for the development of antibiotics that target these bacterial pathogens and also the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. Two distinct pathways form IPP, the mevalonate pathway (Fig. 1Go) and the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway. Wilding et al. (2000) used gene knockouts to establish that the mevalonate pathway represents the sole route to IPP in the Gram-positive cocci. Even more importantly, mevalonate pathway knockout mutants no longer killed infected mice (Wilding et al. 2000). Similarly, fosmidomycin, an inhibitor of the DOXP pathway, protected mice injected with Plasmodium vinckeri (Jomaa et al. 1999), and when administered to children infected with Plasmodium falciparum, yielded encouraging initial results (Borrmann et al. 2004).



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  		Figure 1. The mevalonate pathway for biosynthesis of isopentenyl diphosphate. Three acetyl-CoA units are linked successively by acetoacetyl-CoA thiolase and HMG-CoA synthase to form 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA). Reductive deacylation of HMG-CoA by HMG-CoA reductase forms mevalonate, which undergoes three phosphorylations catalyzed by mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase, forming IPP. Isomerization of IPP to di-methylallyl diphosphate, catalyzed by isopentenyl diphosphate isomerase (data not shown) provides the "building blocks" for the synthesis of all isoprenoids.

 
While these observations clearly establish that blocking IPP synthesis offers potential for antibiotic design and development, the mevalonate pathway is also essential for humans. Consequently, any chemotherapeutic approach targeting this pathway must exploit differences between the bacterial and human enzymes. Such differences indeed exist for at least one enzyme of the pathway. Bacterial and human HMG-CoA reductases exhibit significant differences in three-dimensional structure (Tabernero et al. 1999; Istvan et al. 2000) and their sensitivity to inhibition by the active-site inhibitors known as statins (Tabernero et al. 2003). Comparable differences may also distinguish other bacterial enzymes of the mevalonate pathway from their human counterparts. While the primary structures of the E. faecalis and human enzymes of the pathway generally are <10% identical, the lack of sufficient three-dimensional structures of human and enterococcal members of this pathway preclude detailed comparisons. The structure of a binary complex of mammalian mevalonate kinase (Fig. 1Go) has been solved (Fu et al. 2002), and the coordinates of both human and E. faecalis HMG-CoA synthase (Fig. 1Go), have recently been deposited, and may reveal significant differences.

Phosphomevalonate kinase, the fifth enzyme of the mevalonate pathway, catalyzes transfer of the {gamma}-phosphoryl group of ATP to the hydroxyl of the phosphoryl group of mevalonate 5-phosphate forming mevalonate 5-diphosphate. Phosphomevalonate kinase is a member of the family of GHMP kinases that participate in diverse metabolic processes (Yang et al. 2002) whose shared features include unique kinase folds and a conserved PX3GSSAA sequence that forms a phosphate-binding "p-loop" that contacts the {alpha}-and {beta}-phosphates of ATP (Krishna et al. 2001). Here we report the cloning, expression, purification, and characterization of the phosphomevalonate kinase of E. faecalis.


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 References
 
Multimeric state
The multimeric state of E. faecalis phosphomevalonate kinase was investigated by sedimentation velocity analytical ultracentrifugation (Fig. 2Go). The distribution in Figure 2AGo is uncorrected for solvent density. The corrected values for S20,w and particle mass for the dominant peak at 4.5 S (Fig. 2AGo, peak 2) are 5.0 S and 76.0 kDa. Peak 2 represents 64% of the total mass protein present. Phosphomevalonate kinase thus exists in solution primarily as an observed dimer of 76 kDa (Table 1Go, species 2) which is slightly different from the value of 81 kDa calculated from the sequence.



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  		Figure 2. (A) A c(S) distribution plot of phosphomevalonate kinase. The horizontal axis is not corrected for solvent density and viscosity. Peaks 1 and 2 as labeled in the figure represent molecular weight species 1 and 2 of Table 1Go. (B) Plots of observed fringes (dots) and calculated fringes (lines) against radial position for boundary movement at 15-min intervals. Only every 20th point is shown for the observed data. Data were corrected for radial and time-independent noise effects. (C) The residuals for observed and calculated fringes of B.

 

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  		Table 1. Values of constants calculated from the data in Figure 2BGo
 
Effect of hydrogen ion concentration
The effect of hydrogen ion concentration on the coupling enzymes, pyruvate kinase, and lactate dehydrogenase was determined prior to measuring the effect on phosphome-valonate kinase. Decreased activity above 8.0 was counteracted by additional pyruvate kinase. Subsequent determination of the effect of hydrogen ion concentration on phosphomevalonate kinase activity established that activity occurred over a comparatively broad pH range with optimal activity at pH 8.0 (Fig. S-1; see Supplemental Material).

Effect of temperature
Optimal phosphomevalonate kinase activity occurred at 37°C (Fig. S-2; see Supplemental Material). An activation energy (Ea) of 5.6 kcal/mol was calculated from the slope of 1.22 of an Arrhenius plot of selected data (Fig. S-2, inset; see Supplemental Material) and the relationship slope = Ea/2.303R = Ea/4.58.

Kinetic constants
Km values for phosphomevalonate kinase derived from Lineweaver-Burke plots were 0.19 mM for (R,S)-5-phosphomevalonate (Fig. S-3; see Supplemental Material) and 0.17 mM for ATP (Fig. S-4; see Supplemental Material).

Effect of divalent cations
As anticipated, phosphorylation of phosphomevalonate required a divalent cation. Significantly higher activity was detected using Mn++ than with Mg++. Optimal activity occurred at about 4 mM Mn++ (Fig. 3Go). Ca++, 10 mM, could not replace Mn++ or Mg++.



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  		Figure 3. Divalent cation dependence. Assays of phosphomevalonate kinase activity were conducted at the indicated concentrations of either MnCl2 ({circ}) or MgCl2 (•) under otherwise standard conditions.

 
Specificity of the phosphoryl donor
In preparation for determining the nucleotide specificity of phosphomevalonate kinase, we first investigated the rates at which pyruvate kinase could use nucleoside diphosphates other than ADP. Pyruvate kinase activity was measured by coupling the nucleotide diphosphate-dependent production of pyruvate to the oxidation of NADH catalyzed by lactate dehydrogenase. Relative rates based on four replicate assays for each nucleotide were 100 (ADP), 67 (GDP), 32 (UDP), and 15 (CDP). These rates were sufficient to permit use of the coupled assay to establish which nucleoside triphosphates can serve as phosphoryl donors for phosphomevalonate kinase. Although ATP clearly was preferred, phosphomevalonate kinase utilized phosphoryl donors other than ATP. Relative rates using the indicated nucleoside triphosphates were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP).


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
E. faecalis phosphomevalonate kinase represents the first characterized form of this enzyme from a significant pathogen. Like other members of the GHMP kinase family, it exists in solution primarily as a dimer. Since Mn++ is the preferred cation for phosphoryl group transfer, E. faecalis phosphomevalonate kinase differs from the phosphomevalonate kinases of yeast (Bloch et al. 1959), liver (Levy and Popják 1960), and Streptococcus pneumoniae (Romanowski et al. 2002), and from E. faecalis mevalonate kinase (Hedl and Rodwell 2004), for each of which Mg++ is preferred. As anticipated for an enzyme from a mesophile, activity was optimal at 37°C and decreased sharply at even moderately higher temperatures. It is perhaps not widely appreciated that the coupled assay employed can be used to establish the nucleoside triphosphate specificity of any kinase. This requires only that account be taken of the activity of pyruvate kinase using a given nucleoside diphosphate. In our hands GDP, UDP, and CDP served as phosphoryl acceptors for pyruvate kinase at rates 67%, 32%, and 15% that of ADP. E. faecalis phosphomevalonate kinase was considerably more specific for its phosphoryl donor, where GTP, TTP, and CTP served at only 3.6%, 1.6%, and 0.4% as effectively as ATP.

Antibiotics typically target a reaction unique to the pathogen. That humans and E. faecalis both use exclusively the mevalonate pathway for biosynthesis of IPP does not, however, preclude consideration of this pathway for the design of new antibiotics. Providing that significant structural differences characterize the human and bacterial isoforms, the enzymes of the mevalonate pathway could indeed represent appropriate targets for drug development. But do such differences exist? For HMG-CoA reductase, marked structural differences characterize the class I human enzyme and the class 2 enzyme of a bacterial nonpathogen (Tabernero et al. 2003; Hedl et al. 2004). While at present the only relevant crystal structures are those of HMG-CoA reductase, coordinates are on file for both E. faecalis and human HMG-CoA synthase, and crystallization trials for E. faecalis HMG-CoA reductase have been initiated. The monomers of the bacterial phosphomevalonate kinases are almost twice as large as those of the human and Caenorhabditis elegans. Size differences are most apparent at the N and C termini. Of the three glycine-rich motifs that characterize GHMP kinases (Tsay and Robinson 1991; Zhou et al. 2000), the mammalian and C. elegans enzymes lack motifs 1 and 3, and possibly also motif 2 (Fig. 4Go). Additional differences will become apparent when native and binary complex crystal structures of the pathogen and human enzymes become available. The central metabolic roles of kinases dictate that a useful inhibitor of a specific kinase should be an analog of the substrate rather than of ATP. This parallels the inhibition HMG-CoA reductase by statins, which are analogs of the substrate, not of NADPH.



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  		Figure 4. Sequence alignment of bacterial and eukaryotic phosphomevalonate kinases. Motifs are numbered according to Romanowski et al. (2002). Shading denotes conservation of residues within kingdoms (gray) or across kingdoms (black). S. pyogenes, Streptococcus pyogenes; M. musculus, Mus musculus; R. norvegicus, Rattus norvegicus; H. sapiens, Homo sapiens. Motifs 1, 2, and 3 are also highly conserved in the phosphomevalonate kinases of Saccharomyces cerevisiae and Arabidopsis thaliana.

 
We have now cloned, expressed, purified, and characterized the first five of the six enzymes of the mevalonate pathway in E. faecalis: acetoacetyl-CoA thiolase (Hedl et al. 2002), HMG-CoA synthase (Sutherlin et al. 2002), HMG-CoA reductase (Hedl et al. 2002), mevalonate kinase (Hedl and Rodwell 2004), and now phosphomevalonate kinase. We also recently constructed and operated an immobilized bioreactor composed of the first three E. faecalis enzymes of the mevalonate pathway and described its potential for simultaneous screening of potential inhibitors of several enzymes of a given pathogen (Sutherlin and Rodwell 2004). The cloning, expression, and characterization of E. faecalis phosphomevalonate kinase leaves only the last enzyme of the pathway to complete a bioreactor that incorporates the entire pathway that uses exclusively enzymes from a significant pathogen.


   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Reagents
Purchased materials included Vent DNA polymerase, T4 DNA ligase, and restriction enzymes (New England Biolabs); molecular weight standards and Bradford reagent (BioRad); miniprep kits, gel extraction kits, and Ni-NTA agarose (Qiagen). Chemically synthesized (R,S)-mevalonate 5-phosphate (Wang and Miziorko 2003) was generously provided by Henry Miziorko of the Department of Biochemistry, Medical College of Wisconsin. E. faecalis strain 41 genomic DNA from the GlaxoSmithKline culture collection was a generous gift of Mick Gwynn of GlaxoSmithKline. Synthetic oligonucleotides were synthesized and purified by HPLC by Integrated DNA Technologies, Inc. Other reagents were from Sigma.

DNA manipulations
PCR product purification, plasmid isolation, and DNA extraction from agarose gels kits were from Qiagen. Restriction enzyme-digested DNA was purified using a low melting point agarose gel (Invitrogen) prior to in-gel ligations. Standard protocols (Sambrook et al. 1989) were used for most other DNA manipulations.

Construction of the expression plasmid
E. faecalis strain 41 genomic DNA was used as a template to PCR amplify the 1107-bp mvaK2 open reading frame thought to encode phosphomevalonate kinase. The forward primer 5'-cgcgcgcatatgattgaagttactacg encoded an NdeI site at the N terminus (underlined) and the reverse primer 5'-cgcgcaagctttcatcttttcgattcatgct, a HindIII site at the C terminus (underlined). Ligation into the NdeI and HindIII sites of pET28b(+) (Novagen) yielded expression plasmid pET28EFK2. The DNA sequence of both strands of the insert, determined at the Purdue Genomics Core Facility, corresponded to that of the NCBI database sequence, and verified that no mutation had been introduced during PCR amplification.

Expression and purification of the gene product
Conditions optimal for expression of soluble protein were investigated in small-scale cultures and evaluated by SDS-PAGE. For optimal expression, Escherichia coli BL21(DE3) cells (Invitrogen) transformed with pET28EFK2 were grown initially at 37°C in Luria-Bertani broth (Sambrook et al. 1989) containing 50 µg/mL kanamycin to an A600 of 0.8–1.0, then cooled rapidly to 16°C. Following addition of 0.4 mM IPTG, growth at 16°C was continued for an additional 4–5 h. Cells were harvested by centrifugation; washed in 0.9% saline; suspended in buffer A (300 mM NaCl, 50 mM Tris [pH 8.0]) containing 10 mM imidazole, 1.0 mM phenylmethylsulfonyl fluoride, and 1 µg/mL each of pepstatin, leupeptin, and aprotinin; and ruptured in a French Pressure cell. The supernatant liquid obtained after centrifugation of the cell lysate was applied to a 25-mL column of Ni-NTA agarose equilibrated in buffer A containing 10 mM imidazole and washed in with 50 mL of the same buffer. Elution used successive 50-mL portions of 50 and 100 mM imidazole in buffer A. Migration of the eluted protein on SDS-PAGE (Fig. S-5; see Supplemental Material) was consistent with the observed solution and predicted calculated molecular weights of 48.0 (Table 1Go, species 1) and 40.5 kDa, respectively. Protein yield, determined by the method of Bradford (1976), averaged 20 mg per liter.

MALDI mass spectrometry
Purified protein digested with trypsin was analyzed in positive reflector mode on an Applied Biosystems 4700 MALDI tandem time-of-flight mass spectrometer calibrated with standard peptides (Sigma). A list of monoisotopic peaks from the experimental sample spectra was submitted to Mascot (Matrix Science) for a peptide mass fingerprint analysis using the entire NCBI protein database and a peptide error tolerance of 100 ppm. This returned a single statistically significant result of the nearly two million protein entries in the database, E. faecalis phosphomevalonate kinase. Sequence coverage of the identified protein was 38%.

Assay of phosphomevalonate kinase activity
Measurement of phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple mevalonate 5-phosphate-dependent production of ADP to the oxidation of NADH, monitored spectrophotometrically at 340 nm. Standard assay conditions at 25°C used a 200-µL reaction volume containing 50 mM Tris (pH 8.0), 75 mM KCl, 5 mM MgCl2, 5 mM MnCl2, 2.5 mM phosphoenolpyruvate, 2.0 mM ATP, 1.0 mM (R,S)-phosphomevalonate, 10 mM DTT, ~0.16 mM NADH, 5 units of L-lactate dehydrogenase, and 10 units of pyruvate kinase. Initial rates were calculated from the linear portion of the time course that followed a short initial lag due to the time required to accumulate sufficient ADP and pyruvate. Specific activity is expressed as micromol phosphomevalonate turned over per minute per milligram of protein and was determined to be 3.9 µmol substrate converted per minute per milligram of protein. In experiments where pH or temperature were varied, additional pyruvate kinase and lactate dehydrogenase were included to verify that the activity measured was that of phosphomevalonate kinase, not of the coupling enzymes.

Analytical ultracentrifugation
Sedimentation velocity studies used a Beckman XLI analytical ultracentrifuge and interference optics. Double-sector charcoal-filled epon centerpieces contained 420 µL of 20 mM HEPES (pH 8.0), 300 mM NaCl, and 1.0 mM DTT, plus or minus 1.0 mg/mL enzyme. Density, determined on an Anton-Paar DMA5000 density meter, was 1.016 g/cm3. Values for v (0.7398 mL/g) and viscosity (0.01031p) were calculated from the protein sequence and the buffer composition using SedNterp1.07 (http://www.rasmb.bbri.org/rasmb/windows/sednterp-philo). Following a brief low-speed centrifugation in an An60 Ti rotor at 20°C to sediment insoluble protein and a 60-min equilibration period at 0 rpm, the sample was sedimented at 45,000 rpm for 5 h. Fringe displacement profiles were acquired at 1-min intervals. Analysis of sedimentation velocity profiles used the procedures of Schuck (1998) and Schuck et al. (2002) to refine the positions of the meniscus and cell bottom for the continuous size-distribution analysis. Sedimentation coefficient distributions were calculated with the c(s) method by modeling with distributions of the Lamm equation solutions (Schuck et al. 2002). Analysis of the continuous sedimentation coefficient distribution used Sedfit 8.9 (http://www.analyticalultracentrifugation.com/sedfit.htm). Models with four discrete species were fit to the sedimentation velocity data with Sedphat 2.0b (http://www.analyticalultracentrifugation.com/sedphat/sedphat.htm) using several different weight average masses based on the enzyme sequence for the four largest peaks to insure a global minimum for both s and M.


   Footnotes
 
Supplemental material: see www.proteinscience.org


   Acknowledgments
 
Research support was provided by American Heart Association grant 0150503N to V.W.R. The majority of the data are from the MS thesis of S.D., Purdue University, December 2004. We extend our sincere thanks and gratitude to Timothy Herdendorf and Henry Miziorko for (R,S)-phosphomevalonate and Mark Hall for conducting mass spectrometry. Journal paper 17444 from the Purdue University Agricultural Experiment Station.


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 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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