Biochimica et Biophysica Acta 1784 (2008) 1949–1958

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /bbapap

Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetaseexpressed in Escherichia coli

Venu Kamarthapu a, Khareedu Venkateswara Rao a, P.N.B.S. Srinivas b,G. Bhanuprakash Reddy b, Vudem Dashavantha Reddy a,⁎a Center for Plant Molecular Biology, Osmania University, Hyderabad 500 007, Indiab Biochemistry Division, National Institute of Nutrition, Hyderabad 500 604, India

Abbreviations: SAM, S-adenosylmethionine; ANS, 8-aacid; DTT, dithiothreitol; IPTG, isopropyl β-D-1-thiogalacto⁎ Corresponding author. Fax: +91 40 27096170.

E-mail address: vdreddycpmb@yahoo.com (V.D. Red

1570-9639/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbapap.2008.06.006

a b s t r a c t

a r t i c l e i n f o

Article history:

S-adenosylmethionine (SAM Received 1 May 2008Received in revised form 7 June 2008Accepted 11 June 2008Available online 19 June 2008

Keywords:Bacillus subtilisHeterologus hostmetES-adenosylmethionine synthetaseUrea-induced unfolding

) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionineusingL-methionine and ATP as substrates. SAM synthetase gene (metE) from Bacillus subtilis was clonedand over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~180 kDa, suggesting that the enzymeis a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity atpH 8.0 and 45 °C with the requirement of divalent cation Mg2+, and stimulated by the monovalentcation K+. The enzyme followed sequential mechanism with a Vmax of 0.362 µmol/min/mg, and a Km of920 µM and 260 µM for ATP andL-methionine, respectively. The urea-induced unfolding equilibrium ofthe recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer,structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsicand extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates thatthe enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in thepresence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzymeactivity.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) mediatessynthesis of S-adenosylmethionine using L-methionine and ATP assubstrates [1]. Synthesis of SAM occurs in an unusual two-stepreaction, in which the triphosphate chain is cleaved from ATP as SAMis formed, and the triphosphate is further hydrolyzed to PPi and Pibefore the sulfonium product is released [2]. SAM synthetase uniquelycatalyzes reactions at both ends of the tripolyphosphate chain in adefined temporal sequence [3].

SAM is widely used as a methyl donor, and many essentialfunctions of the cell are dependent on methylation reactions. Duringmethyl transfer, SAM is converted to S-adenosylhomocysteine andsubsequent hydrolysis forms adenosine and homocysteine. Homo-cysteine in turn is either converted to glutathione, a major cellularantioxidant, or methylated to generate methionine [4,5]. SAM is alsoused as a source of methylene groups involved in the synthesis ofcyclopropyl fatty acids, amino groups for synthesis of epoxyqueuosine

nilinonaphthalene-1-sulphonicpyronoside

dy).

l rights reserved.

and aminopropyl groups involved in synthesis of ethylene andpolyamines [6].

SAM is extensively used in the treatment of depression, dementia,vacuolar myelopathy, liver injury, migraine, osteoarthritis [5], and as apotential cancer chemopreventive agent [7]. Certain genes mediatingSAM metabolism have been found to serve as important drug targetsfor development of chemotherapeutic agents against neoplastic andviral diseases [8]. Because of cardinal manifestations of SAM, SAMsynthetase acquires immense significance.

SAM synthetase from Escherichia coli [9], Saccharomyces cerevi-siae [10], Sulfolobus solfataricus [11], Leishmania infantum [12], Rat-tus norvegicus [13] and Homo sapiens [14] has been purified andwere found to show low specific activity. To date crystal structure oftetrameric SAM synthetases from E. coli [3], R. norvegicus liver-specific enzyme MAT I [15] and human MAT I and MAT II [PDBcodes 2obv, 2p02, unpublished] have been determined. In mam-mals, two isoforms have been reported, one ubiquitous MAT II andother liver-specific enzyme that can exist as homotetramer MAT Iand homodimer MAT III [16]. The in vivo oligomerization state ofMAT III dimer is widely documented in the literature, but no crystalstructure determination of this form has been reported until now[16]. Overproduction of SAM synthetase in Bacillus subtilis leads tomethionine auxotrophy [17]. Many aspects of the unusual two-stepenzymatic mechanism of SAM synthetase remain unknown and,

1950 V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

therefore, more experimental data from this interesting family ofenzyme is needed to get a deeper insight into its function. In viewof variation in structure and low specific activity of the enzyme,characterization of SAM synthetases from other organisms isdesirable.

In spite of numerous studies, protein folding remains as one ofthe challenging areas of structural biology [18,19]. Characterizationof folding intermediates is considered as an important strategy forelucidating of the mechanism of protein folding. Multidomain andoligomeric proteins remain relatively little explored [20]. Folding ofoligomeric proteins follows two- to five-state processes involvingmonomeric and/or multimeric intermediates [21,22]. Associationsmight occur among completely folded monomers or, alternatively,final folding occurs after association of partially folded subunits[23]. Since folding/unfolding of such proteins involve association/dissociation of subunits, these processes seem much morecomplicated than that of monomeric proteins, and such studiesmight provide additional insights into the interdependence offolding and oligomerization processes [24]. To understand themechanisms of folding and assembly of oligomeric proteins, it isessential to characterize the equilibrium unfolding intermediates atdifferent stages of unfolding as well as the relationship betweenthem.

In this study, we dealt with the cloning of SAM synthetase genefrom B. subtilis, and its functional overexpression in the heterologushost E. coli. The recombinant enzyme has been purified andcharacterized for various properties, viz., optimal pH, temperature,metal dependency and kinetic parameters for its substrates. We alsoinvestigated urea-induced unfolding of the recombinant enzyme andintermediate states present in the unfolding process, which weremonitored by intrinsic fluorescence, near and far-UV CD spectra, SECand binding with the extrinsic hydrophobic fluorescence probe ANS,besides establishing the structure–function relationship.

2. Materials and methods

2.1. Chemicals and reagents

ATP, methionine, ANS (8-anilinonaphthalene-1-sulphonic acid),dithiothreitol, and urea were obtained from Sigma (St. Louis, USA).Whereas, all other chemicals were of highest purity procured fromlocal companies.

2.2. Amplification of metE gene and its expression in E. coli

Genomic DNA was purified from B. subtilis [25]. The metE genewas amplified by PCR with forward 5′ CATATGAGCAAAAATCGTCGTT-TATTTACATC 3′ and reverse 5′ CGAGAATTCCTATTCTCCTAACGCTTCTT-TAC 3′ primers containing Nde I and EcoR I sites respectively. Theamplified fragment was cloned in to Sma I site of pBluescript KS (+)(Stratagene, La Jolla, CA, USA). The SAM synthetase gene frompBluescript KS (+) was excised by Nde I and EcoR I and subcloneddown stream T7 promoter in frame with 6X His-tag of pET28a (+)expression vector, resulting construct pETSAM was confirmed bysequencing both strands. The pETSAM plasmid was introduced in toE. coli BL21 (DE3) [26]. Transformed cells were grown in 100 ml of LBmedium containing 50 µg of kanamycin/ml in 500 ml flask at 37 °Cand 250 rpm until the cell density reached an A600 nm of 0.6 to 0.8.The culture was then cooled to 20 °C and IPTG was added to a finalconcentration of 50 µM. Cells were grown for additional 2 h at 20 °Cand 100 rpm. Cells from 500 ml induced culture were harvested bycentrifugation (8000 ×g for 15 min) at 4 °C. The pellet wasresuspended and washed three times in ice-cold phosphate bufferedsaline (PBS) solution. The cells were resuspended in 50 ml of 50 mMsodium phosphate buffer (pH 8.0) containing 300 mM NaCl, 10 mMimidazole and 10% glycerol (lysis buffer) and lysed by sonication 10 s

thrice at 50% level and cell debris was cleared by centrifugation. Theexpression of the SAM synthetase was checked by 12% SDS-PAGE[27].

2.3. Purification of recombinant SAM synthetase

The cell lysate was loaded on to Ni-NTA column with 4.0 ml ofbed volume and washed successively with 50 ml of lysis buffer,100 ml of 50 mM sodium phosphate buffer (pH 8.0) containing300 mM NaCl, 20 mM imidazole and 10% glycerol (wash buffer).The bound SAM synthetase was eluted out of the Ni-NTA columnusing 10 ml of 50 mM sodium phosphate buffer (pH 8.0)containing 300 mM NaCl, 250 mM imidazole and 10% glycerol.Further, SAM synthetase was subjected to buffer (100 mM Tris–Cl,pH 8.0 containing 10% glycerol) exchange by PD-10 column (GEHealth Care, UK). For long term storage glycerol was added to afinal concentration of 20% and stored at −70 °C. The homogeneityof the purified protein was assessed using 12% SDS-PAGE. His-tagged enzyme was digested with thrombin to remove 6× His-tag.No changes were observed in the fluorescence, CD spectra,oligomeric state and activity of the enzyme with and withoutHis-tag. Hence, all further assays were carried out using the His-tagged recombinant enzyme. Protein content of samples wasdetermined by the method of Bradford [28] using BSA asstandard.

2.4. Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was performed using IPGstrips (7 cm, pH 3–10; Bio-Rad, Hercules, CA, USA) for IEF in the firstdimension followed by SDS-PAGE in 12% acrylamide in the seconddimension. IPG strips were rehydrated passively with rehydrationbuffer (8 M urea, 2% CHAPS, 50 mM DTT and 0.2% carrier ampholytespH 3–10; Bio-Rad) premixed with 20 µg of the sample. IEF wascarried out in a Protean IEF cell (Bio-Rad) with end voltage of 4000 Vat 20 °C for a total of 10,000 Vh. Gel was stained with CoomassieblueR-250.

2.5. SAM synthetase activity assay

SAM synthetase activity was assayed essentially as per previouslyreported methods [29,30], with minor modifications. Unless andotherwise stated the standard reaction mixture containing 100 mMTris–Cl pH 8.0, 20 mM MgCl2, 200 mM KCl, 5 mM ATP, and 5 mML-methionine in a final reaction volume of 1.0 ml with 0.05 mg ofenzyme was incubated at 37 °C for 2 h. The reaction was stopped byaddition of 1.0 ml of cold 10% HClO4. A blank control was preparedsimultaneously without ATP. The formation of SAM was analyzed bya high performance liquid chromatography (HPLC) (Shimadzu,Japan) using a Hypersail SCX column (4.6 mm×250 mm, 5 µm).The mobile phase consist of 100 mM ammonium formate (pH wasadjusted to 4.0 with formic acid). The formation of SAM wasmonitored at λ=254 nm. SAM was identified and quantified byknown concentration of the standard compound SAM (Sigma, USA).One unit of the enzyme activity was defined as the formation of1 µmol of SAM in 1 min in the standard assay mixture. The datawere analyzed by the Sigma Plot, Enzyme Kinetics Module (SYSTATSoftware Inc., Chicago, IL).

2.6. Effect of temperature, pH and cations on enzyme activity

To determine pH optima, defined buffers viz., pH 4.0–6.0 (100 mMcitrate buffer), pH 7.0–9.0 (100 mM Tris–Cl), and pH 10.0 (100 mMcarbonate buffer) were used under standard assay conditions. Optimaltemperature of the enzymatic activity was determined under standardassay condition by incubating at varying temperatures from 25 °C to

1951V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

55 °C. Effect of monovalent ions (K+, Li+, Na+ and NH4+) at 200 mM and

divalent ions (Mg2+, Ca2+, Co2+, Ni2+ and Zn2+) at 20mMon the enzymeactivity was estimated under standard reaction conditions.

2.7. Characterization of the enzyme for its kinetic constants

To determine the kinetic constants of the enzyme, the purifiedSAM synthetase was assessed under standard reaction conditions atvarious ATP concentrations (0–3.0 mM) and fixed L-methionineconcentration and varied L-methionine concentrations (0–4.0 mM)and fixed ATP concentration.

2.8. Protein denaturation

SAM synthetase denaturation experiments with varied concentra-tions of urea were carried out in 10 mM Tris–Cl (pH 8.0) at 25 °C. Toensure equilibrium state, samples were incubated at least for 2 h andused for recording measurements.

2.9. Circular dichroism (CD) analysis

CD spectra were recorded as an average of six accumulations atroom temperature using a Jasco J-810 spectropolarimeter. SAM

Fig. 1. Deduced amino acid sequence of SAM synthetase from Bacillus

synthetase equilibrated with different concentrations of urea (0 to5.0 M) in 10 mM Tris–Cl buffer (pH 8.0) were used for recording far-and near-UV CD spectra at room temperature employing cells of 0.2and 0.5 cm path length, respectively. Spectra were corrected withrespective blanks. Protein concentrations used for far- and near-UVwere 0.1 and 1.0 mg/ml, respectively. The CD data are expressed asmolar ellipticity (deg.cm2.dmol−1).

2.10. Fluorescence measurements

Tryptophan fluorescence emission spectra (300–400 nm) ofSAM synthetase were recorded as a function of urea concentra-tion (0–5.0 M) by excitation at 295 nm using 0.1 mg/ml proteinconcentration. Fifty µM ANS with 0.1 mg/ml either native or ureadenatured SAM synthetase in 10 mM Tris–Cl buffer (pH 8.0) wasincubated for 30 min at room temperature in dark. The mixturewas subjected to excitation at 390 nm and fluorescence emissionspectra of protein-bound dye were recorded between 400 and600 nm. Background fluorescence due to buffer, ANS, urea andprotein were corrected using appropriate blanks. All measure-ments were recorded at 25 °C on Jasco FP-6500 spectrofluoro-meter using 3 nm spectral bandwidths for both excitation andemission.

subtilis exhibiting homology with the enzyme from other genera.

1952 V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

2.11. Size-exclusion chromatography

To monitor the urea-induced quaternary structural change, theenzymewas subjected to SEC. Hundred µg of SAM synthetase in 100 µlof 10 mM Tris–Cl (pH 8.0) with varying urea concentrations wereincubated at room temperature for 2 h. Samples were loaded on to a300×7.5 mm TSK-G3000SWxl column (Tosoh, Tokyo, Japan) con-nected to a Shimadzu HPLC system, at a flow rate of 1 ml/min. Thecolumnwas equilibrated and proteins were elutedwith 50mMTris–Clbuffer (pH 8.0) containing 50 mM KCl, 1 mM EDTA, 0.1% β-mercaptoethanol and 10% glycerol, including an appropriate concen-tration of urea. Thyroglobulin (669 kDa), catalase (240 kDa), gammaglobulin (150 kDa), BSA (66 kDa), ovalbumin (45 kDa) and ribonu-clease A (29 kDa) were used as standards.

2.12. Effect of urea on enzyme activity

The purified enzyme (0.1 mg) was incubated in 100 mM Tris–Cl(pH 8.0) with varying concentration of urea (0–5.0 M) at roomtemperature. After 2 h, the samples were assayed for enzyme activityand compared with that of controls.

3. Results and discussion

SAM acts as a major methyl donor in biological systems, besides itsrole in processes such as synthesis of polyamines [31], radical-based

Fig. 2. Expression, purification and determination of pI of Bacillus SAM synthetase. (Panel A) Sinduced E. coli harbouring pETSAM showing expression of recombinant protein; Lane 3.Molecular weight marker. (Panel B) The scale given above electrophoretogram indicates themarker spot displayed at pI 7.0.

catalysis [32] and ethylene synthesis in plants [6]. In addition to itsrole inmetabolism, SAM is also involved in the regulated expression ofS-box genes [33] andmorphological differentiation of prokaryotes andeukaryotes [34,35]. This investigation deals with cloning of B. subtilisSAM synthetase gene (metE) and its overexpression in active form in E.coli system. The recombinant enzyme was further analyzed for itscatalytic and structural properties besides urea-induced unfolding.

3.1. Cloning and characterization of SAM synthetase

Gene (metE) coding for SAM synthetase, containing 1203 nucleo-tides, was cloned from B. subtilis and it encoded a protein of 400amino acids (Fig. 1). The deduced amino acid sequence of B. subtilisSAM synthetase revealed an identity of 61% with that of E. coli, 54%with S. cerevisiae, 58% with human and 57% with rat enzyme, thusindicating the highly conserved nature of the enzyme. The amino acidsequence contained cofactor binding sites of D31 and D286 for Mg2+

and E45 for K+ including a conserved motif P-loop HGGGAFSGKD(272–281 amino acids), as well as the signature hexa peptide GAGDQG(129–135 amino acids), signify evolutionarily conserved nature of theenzyme across species and genera.

3.2. Expression of soluble and active SAM synthetase

E. coli BL 21 (DE3), harbouring B. subtilis metE gene downstream toT7 promoter, when induced with 1 mM IPTG at 37 °C and 225 rpm

DS-PAGE. Lane 1. Lysate of uninduced E. coli harbouring pETSAM; Lane 2. Lysate of IPTG-Nickel (Ni2+)-affinity chromatography purified recombinant SAM synthetase; Lane 4.pH gradient in the first dimension. SAM synthetase spot corresponds to pI 5.2. Standard

Fig. 3. Effect of pH and temperature on enzyme activity of SAM synthetase. (Panel A)The enzyme assay was performed in buffer with varying pH (4–10) at 37 °C for 2 h.(Panel B) Enzyme assay was performed at different temperatures (25–55 °C) at pH 8.0for 2 h. Data represent Mean±S.E. for three assays.

Fig. 4. Double reciprocal plots of the SAM synthetase. The standard kinetic reactionmixture is described in the text. In each case the slope of the plot decreased withincreasing concentration of substrate. (Panel A) L-methionine was varied at fixed ATPconcentrations of 0.5, 1.0, 2.0 and 3.0 mM. (Panel B) ATP was varied at fixed L-methionine concentrations of 0.5, 1.0, 2.0 and 4.0 mM. Results shown are average ofthree independent assays.

1953V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

resulted in the formation of inactive inclusion bodies. Purifiedinclusion bodies were dissolvedwith chaotropic agent urea/guanidineHCl and used for refolding. Several attempts made to refold theenzyme failed to yield the active enzyme. However, induction of E. coliwith 50 µM of IPTG, at 20 °C and shaking (100 rpm) was foundoptimum for overexpression of recombinant SAM synthetase in activesoluble form. Formation of inactive inclusion bodies at 37 °C, 225 rpmupon induction with IPTG (1.0 mM) suggests active metabolic state ofthe recombinant E. coli with enhanced transcription and translationalactivities. The ambient temperature (37 °C) might be favouring strongintra- and/or inter-molecular hydrophobic interactions resulting in animproper folding and aggregation of the recombinant protein.Conversely, low concentration of inducer (50 µM IPTG) and sub-optimal culture conditions, viz., temperature (20 °C) and shaking(100 rpm), might have favoured decreased metabolic activity,transcription and translation, resulting in an active soluble enzyme.Further, reductions in culture temperature were found to increaseexpression and activity of E. coli chaperones [36,37], therebyfacilitating proper folding of SAM synthetase in the heterologushost. In earlier studies, increased amount of soluble proteins wererecovered owing to low-level transcription from moderate/weakpromoters as well as reduced activity of strong promoters [38].

The purified protein showed a single band of ~45 kDa (Fig. 2A) onSDS-PAGE under reducing conditions confirming the purity of protein;two-dimensional electrophoresis further revealed pI of ~5.2 (Fig. 2B),which correlated well with that of theoretical values. As compared toglycerol free buffers, supplementationwith glycerol (10%) contributedto increased recovery and stability of the active enzyme. Glycerol is

well known for its favourable influence on the structural integrity ofproperly folded proteins besides its destabilizing effect on relativelyunfolded forms [39]. Size-exclusion chromatography revealed a singlepeak corresponding to ~180 kDa (Fig. 9), indicating that the nativeSAM synthetase might be a homotetramer. Oligomeric nature of SAMsynthetase was earlier reported in E. coli as a tetramer [3], in rat astetramer (MAT I) and dimer (MAT III) [15,16] with intersubunit activesite(s) [40], and as a dimer in S. cerevisiae [2].

3.3. Effect of pH, temperature and cations on enzyme activity

The enzyme was found active between pH 6.0 and pH 10.0 withmaximum activity at pH 8.0 (Fig. 3A). Similar optimal pHwas reportedfor SAM synthetases of E. coli and yeast [2,9]. Although the enzymeexhibited measurable activity at various temperatures ranging from25 °C to 55 °C, it showed peak activity at 45 °C (Fig. 3B). However, SAMsynthetases from other species were reported to have an optimumtemperature of ~30 °C. The recombinant enzyme showed no activity atpH 5.0; its inactivity at lower pHmay be attributed to the alterations inthe charge of amino acids that are involved in metal binding. Thedivalent ion requirement of the SAM synthetase was satisfied by Mg2+

with a maximal relative activity of 1.0 followed by Ca2+ (0.79), Co2+

Fig. 5. Intrinsic tryptophan fluorescence of SAM synthetase at varying concentrations ofurea. SAM synthetase (0.1 mg/ml) was incubated at 0–5.0 M urea for 2 h at roomtemperature before fluorescence measurements were made. (Panel A) Intrinsictryptophan fluorescence of SAM synthetase at various concentrations of urea. Curves1–6 represent the spectra of the protein at 0, 0.3, 0.8, 1.4, 2.8 and 5.0 M urea,respectively. (Panel B) Plots of emission maximum as a function of urea concentration.(Panel C) Relative fluorescence emission intensity at 336 nm (au: arbitrary units).

Fig. 6. ANS-associated fluorescence of SAM synthetase as a function of urea. SAMsynthetase (0.1 mg/ml) of native/urea (0–5.0 M) denatured enzyme was incubated with50 µM of ANS at room temperature in dark for 30 min and fluorescence intensity wasrecorded. (Panel A) Curves 1–6 represent the spectra of protein at 0, 0.6, 1.4, 1.8, 2.4 and5.0 M urea respectively. (Panel B) Relative change in ANS fluorescence at 470 nm as afunction of urea concentration.

1954 V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

(0.59), Ni2+(0.47) and Zn2+ (0.08). Similarly, a maximal relative activityof 1.0 was achieved with the monovalent K+ ion followed by Li+ (0.77),NH4

+ (0.52) and Na+ (0.36). Addition of EDTA to the reaction mixresulted in complete inhibition of enzyme activity, indicatingrequirement of divalent metal ion for its activity. Further, the presenceof conserved amino acids at positions D31 and D286 for Mg2+ bindingsites and E45 for the K+ binding site authenticate the need for bothdivalent and monovalent ions for enzyme activity. Presence of twodivalent ions and one monovalent ion in each subunit were found atthe active site of E. coli SAM synthetase [3], and rat MAT I enzyme [15].

Stability of the SAMwas tested by incubating for 3 h in the varying pH(4–10) and temperatures (25 °C–55 °C) and was found stable.

3.4. Steady state kinetic mechanism of SAM synthetase

Activity assay was performed to determine the kinetic constants ofSAM synthetase. The enzyme followed Michaelis–Menten kinetics.Lineweaver–Burk plot analysis revealed a Vmax of 0.362 µmol/min/mgprotein, and a Km of 920 µM and 260 µM for ATP and L-methionine,respectively (Fig. 4A, B). Plot of 1/v versus 1/s with L-methionine/ATPas variable substrate(s) resulted in straight lines intersecting to the leftof vertical axis, thus suggesting a sequential mechanism in whichbinding of both substrates is essential for release of the product.

3.5. Urea-induced unfolding of SAM synthetase

Study of equilibrium unfolding of proteins reveals differentfeatures influencing the conformational stability as well as mechan-isms involved in the folding of globular proteins. The unfolded state ofa protein represents a random conformation with a high degree ofsolvent exposure besides flexibility of side chains and the backbone[41–43].

3.5.1. Intrinsic tryptophan fluorescenceA single tryptophan residue at position 384 was used as a probe to

determine the change in fluorescence intensity as a function of urea

Fig. 7. Urea-induced unfolding of SAM synthetase monitored by far-UV CD spectra.(Panel A) Curves 1 to 7 represent spectra of the protein at 0, 0.8, 2.0, 2.8, 3.6 and 5.0 Murea. (Panel B) Variation of molar ellipticity at 222 nm as a function of ureaconcentration.

1955V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

concentration. The native enzyme exhibited emission maximum(λmax) at 331 nm. With increasing concentration of urea, a red shiftin emission was detected which plateaued at 356 nm in 5.0 M urea(Fig. 5A, B). Relative fluorescence intensity at 336 nm increased withincreasing concentration of urea and was found maximum at 0.8 Murea (Fig. 5C), indicating that the location of tryptophan in the nativeprotein might be in the weak hydrophobic region and its probabletransition to an unexposed state in the partially unfolded enzyme.Further increase in the concentration of urea resulted in a concomi-

Fig. 8. Urea-induced unfolding of SAM synthetase monitored by near-UV CD spectra.Curves 1 to 4 represent spectra of the protein at 0, 0.4, 1.0 and 2.0 M urea, respectively.

tant decrease in the fluorescence intensity owing to increasedexposure of tryptophan, leading to complete unfolding of enzyme at2.8 M urea [44]. As compared to the native enzyme, denatured proteinat 1.0 M urea revealed two-fold increase in fluorescence intensity (Fig.5A), plausibly caused by changes in the microenvironment of thetryptophan at 384 amino acid position.

Increasing concentration of urea exhibited a profile of λmax,indicative of non-two-state transition with an inflection point at

Fig. 9. Elution profiles of SAM synthetase at different concentrations of urea weremonitored on a TSK _G 3000SWxl Gel filtration HPLC column. SAM synthetase in bufferalone (trace 1), buffer containing 0.4 M urea (trace 2), 0.75 M urea (trace 3), 1.0 M urea(trace 4), and 2.0 M urea (trace 5). Elution positions of standard molecular-massmarkers. A, catalase (240 kDa); B, gamma globulin (150 kDa); C, ovalbumin (45 kDa); D,ribonuclease A (29 kDa).

Fig. 10. Enzyme activity of SAM synthetase at various concentrations of urea. SAMsynthetase (0.1 mg) was incubated at 0 to 5.0M urea for 2 h at room temperature (25 °C)prior to enzyme assay. Results shown are average of three independent assays.

1956 V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

~1.0 M urea (Fig. 5B). Changes in fluorescence profiles of denaturedenzyme, consequent to alterations in the microenvironment ofchromophore tryptophan, suggest the probable existence of morethan one intermediate state in the unfolding pathway between 0.4 Mand 1.4 M urea. Increase in intrinsic fluorescence intensity up to 0.8 Murea followed by gradual decrease in fluorescence intensity withfurther increasing concentrations of denaturant indicates plausibleexistence of more than one intermediate state in the proteindenaturation pathway. Similar changes were also observed in othermultimeric proteins [45,46].

3.5.2. Binding of ANS to the enzymeThe binding of ANS to SAM synthetase in the presence of various

concentrations of urea was recorded using fluorescence spectra from400 to 600 nm (Fig. 6A, B). SAM synthetase with increasingconcentration of urea up to 1.4 M showed marginal increases in ANSfluorescence intensity (at 470 nm) owing to partial unfolding andexposure of intersubunit hydrophobic surfaces, resulting in enhancedbinding of ANS. However, further increase in urea concentrationdecreased fluorescence intensity and became negligible at 5.0 M,indicating extreme unfolding of the enzyme. Earlier studies with ratmethionine adenosyl transferase revealed increased ANS fluorescenceat lower concentration of the denaturant, while a drastic decrease wasreported at higher concentration of the denaturant; the changesobserved in the fluorescence were ascribed to dissociation of dimerand exposure of intersubunit apolar regions followed by completedenaturation of monomers [16].

Fig. 11. Model representing the urea-induced equilib

3.5.3. Unfolding of enzyme monitored by CD spectraFar-UV CD spectral studies on urea-induced unfolding of SAM

synthetase were carried out to investigate the effect of urea onsecondary structure of the protein. In far-UV region, the CD spectrumof native protein revealed presence of 81%α-helix, 8% β-sheet and 11%random coil content, as determined by the CDpro software (http://lamar.colostate.edu/~sreeram/CDPro/main.shtml). Increasing concen-tration of urea showed decreased molar ellipticity of the protein (Fig.7A). Native protein disclosed two prominent negative bands at 208and 222 nm (Fig. 7A). Molar ellipticity at 222 nm also decreased withincreasing concentration of urea. Up to 2.0 M urea, minute decreasesin molar ellipticity were recorded, and from 2.0 to 3.5 M ureaprominent decreases in molar ellipticity were observed (Fig. 7B).Negligible changes observed in molar ellipticity up to 1.4 M ureapoints to the stable maintenance of secondary structure. Far-UV CDspectra revealed retention of substantial component of secondarystructure even at 2.0 M urea. A gradual increase in molar ellipticitywith further increases in urea concentration indicates gradual loss ofsecondary structure. Negligible amount of molar ellipticity observedat 5.0 M urea suggests complete unfolding of the secondary structure.

Near-UV CD spectra of native SAM synthetase showed a positiveband at 295 nm due to tryptophan and a negative band at 275 nmregion as a combined effect of both tryptophan and tyrosine. Upondenaturation the enzyme showed an altered signal even at 0.4 M urea.However, the protein showed no ellipticity at 2.0 M urea (Fig. 8),suggesting that unfolding intermediates might develop loosened side-chain packing and loss of tertiary structure with further denaturation[47].

3.5.4. Size-exclusion chromatographyChanges in oligomeric state of the protein in presence of varying

concentration of urea were determined subjecting to SEC. SAMsynthetase in its native conformation was eluted as a single peakcorresponding to a molecular weight of ~180 kDa and disclosedsimilar elution profile at 0.25 M urea, indicates the retention ofoligomeric state with plausible minor changes. At 0.4 M urea, increasein elution time resulted in a peak corresponding to dimer (~90 kDa)and a shoulder of monomer (~45 kDa). By increasing the concentra-tion of urea to 0.75 M, increases in the fraction of monomer with acorresponding decrease in the dimer were observed as evidenced byelution profile (Fig. 9). Existence of a population of monomeric anddimeric molecules points to an equilibration between dimeric andmonomeric species at 0.40–0.75 M urea. Elution profile from 1.0 Murea onwards showed a single peak corresponding to the monomer,indicating complete loss of quaternary structure. Unfolding profiles inSEC suggest the tetrameric nature of Bacillus SAM synthetase. Absenceof the peak corresponding to trimer in the SEC, revealed that

rium unfolding of SAM synthetase of B. subtilis.

1957V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

tetrameric Bacillus SAM synthetase results from the assembly of twohomodimeric molecules and not due to sequential addition ofmonomers. Glutaraldehyde cross-linking of the protein displayedbands corresponding to tetramer, dimer and monomer but withoutany trimer band (data not shown), further supports that the enzyme isa dimer of dimer. These results are in agreement with earlier studieswherein the enzyme has been reported as a dimer of dimer [3,15].However, rat MAT III was found as dimer [48].

3.5.5. Enzyme activityTo establish the relationship of enzyme activity with that of

intermediates in the unfolding pathway, enzyme assays were carriedout in the presence of various concentrations of urea (0–5.0 M). Lossof ~50% activity was observed at 0.2 M urea, whereas 0.4 M ureashowed complete loss of enzyme activity (Fig. 10). Total loss of en-zyme activity and absence of tetramer in size-exclusion chromato-graphy, at 0.4 M urea, authenticate that the tetrameric form alone isactive while lower forms lack enzyme activity. A notable decrease inenzyme activity at 0.2 M urea, despite maintenance of tetramericform, is attributable to an altered conformation, as evidenced byincrease in intrinsic fluorescence. Alteration in enzyme conformationmight have resulted from a combination of various factors, viz.,changes in microenvironment of active site leading to reduction inbinding affinity for substrate and cofactor, change(s) in hydrophobicand hydrophilic domains, altered tertiary structure, etc. These resultsalso indicate that the active site of enzyme is probably located in theintersubunit region, and can be easily disturbed at low concentra-tions of urea since interaction between subunits are non-covalentand predominantly hydrophobic. The SDS-PAGE revealed thatmonomeric state of enzyme is attained in the absence of reducingagents like DTT or β-mercaptoethanol, implicating lack of inter-subunit disulphide linkages.

Based on accrued results, we propose a representative model ofurea-induced-equilibrium unfolding pathway for Bacillus SAMsynthetase (Fig. 11). Unfolding of homotetramer proceeds throughtwo distinct stages, a pre-denaturation state and a global unfoldingstate. In the first stage, at 0.2 M urea, there is no alteration in thetetrameric state but lose ~50% of its activity while retaining tertiaryand secondary structure. Increase in urea concentration up to 0.4 M,reduces quaternary status of the enzyme dissociating into dimer withan altered tertiary structure thereby loosing enzyme activity. In thesecond stage, at N0.75 M urea, dimers dissociate into monomers andlose substantial tertiary structure while retaining secondary structure.Further increase in urea concentration (1.0 to 5.0 M) promotes a broadunfolding transition wherein unfolding monomeric intermediates areconverted to completely unfolded monomers.

In conclusion, B. subtilis metE gene upon overexpression in E. coli atsub-optimal culture conditions yielded soluble, functional SAMsynthetase. The overall results of equilibrium unfolding, size-exclu-sion chromatography and enzyme activity, first of its kind, revealedthat the active form of enzyme is a homotetramer stabilized by non-covalent interactions. The unfolding pathway of the enzyme proceedsto monomer through dimer, thereby establishing that B. subtilis SAMsynthetase is a dimer of dimer with an active site in the intersubunitregion.

Acknowledgements

We extend our thanks to Professor T. Papi Reddy, Former Head,Department of Genetics, Osmania University, Hyderabad, for thehelpful suggestions and evaluation of the manuscript. We thank Prof.T. N. Raju, Department of Zoology, Osmania University, Hyderabad, forextending the HPLC facility. We also acknowledge the help renderedby Mr. Rajinikanth. Financial support from the Department ofBiotechnology, Government of India (New Delhi) is gratefullyacknowledged.

References

[1] G.L. Cantoni, S-adenosylmethionine; a new intermediate formed enzymaticallyfromL-methionine and adenosinetriphosphate, J Biol Chem 204 (1953) 403–416.

[2] S.H. Mudd, G.L. Cantoni, Activation of methionine for transmethylation. III. Themethionine-activating enzyme of Bakers' yeast, J Biol Chem 231 (1958) 481–492.

[3] F. Takusagawa, S. Kamitori, S. Misaki, G.D. Markham, Crystal structure ofS-adenosylmethionine synthetase, J Biol Chem 271 (1996) 136–147.

[4] P.K. Chiang, R.K. Gordon, J. Tal, G.C. Zeng, B.P. Doctor, K. Pardhasaradhi, P.P.McCann, S-adenosylmethionine and methylation, FASEB J 10 (1996) 471–480.

[5] T. Bottiglieri, S-adenosyl-L-methionine (SAMe): from the bench to the bedside-molecular basis of a pleiotrophic molecule, Am J Clin Nutr 76 (2002) 1151S–1157S.

[6] M. Fontecave, M. Atta, E. Mulliez, S-adenosylmethionine: nothing goes to waste,Trends Biochem Sci 29 (2004) 243–249.

[7] R.M. Pascale, V. Marras, M.M. Simile, L. Daino, G. Pinna, S. Bennati, M. Carta, M.A.Seddaiu, G. Massarelli, F. Feo, Chemoprevention of rat liver carcinogenesis byS-adenosyl-L-methionine: a long-term study, Cancer Res 52 (1992) 4979–4986.

[8] A.E. Pegg, Polyamine metabolism and its importance in neoplastic growth and atarget for chemotherapy, Cancer Res 48 (1988) 759–774.

[9] G.D. Markham, E.W. Hafner, C.W. Tabor, H. Tabor, S-adenosylmethioninesynthetase from Escherichia coli, J Biol Chem 255 (1980) 9082–9092.

[10] P.K. Chiang, G.L. Cantoni, Activation of methionine for transmethylation.Purification of the S-adenosylmethionine synthetase of bakers' yeast and itsseparation into two forms, J Biol Chem 252 (1977) 4506–4513.

[11] M. Porcelli, G. Cacciapuoti, M. Carteni-Farina, A. Gambacorta, S-adenosylmethio-nine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.Purification and characterization of two isoforms, Eur J Biochem 177 (1988)273–280.

[12] R.M. Reguera, R. Balana-Fouce, Y. Perez-Pertejo, F.J. Fernandez, C. Garcia-Estrada,J.C. Cubria, C. Ordonez, D. Ordonez, Cloning expression and characterization ofmethionine adenosyltransferase in Leishmania infantum promastigotes, J BiolChem 277 (2002) 3158–3167.

[13] D.M. Sullivan, J.L. Hoffman, Fractionation and kinetic properties of rat liver andkidney methionine adenosyltransferase isozymes, Biochemistry 22 (1983)1636–1641.

[14] M. Kotb, N.M. Kredich, S-adenosylmethionine synthetase from human lympho-cytes. Purification and characterization, J Biol Chem 260 (1985) 3923–3930.

[15] B. González, M.A. Pajares, J.A. Hermoso, L. Alvarez, F. Garrido, J.R. Sufrin, J.Sanz-Aparicio, The crystal structure of tetrameric methionine adenosyltrans-ferase from rat liver reveals the methionine-binding site, J Mol Biol 300 (2000)363–375.

[16] M. Gasset, C. Alfonso, J.L. Neira, G. Rivas, M.A. Pajares, Equilibrium unfoldingstudies of the rat liver methionine adenosyltransferase III, a dimeric enzyme withintersubunit active sites, Biochem J 361 (2002) 307–315.

[17] R.R. Yocum, J.B. Perkins, C.L. Howitt, J. Pero, Cloning and characterization of themetE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis,J Bacteriol 178 (1996) 4604–4610.

[18] T.E. Creighton, Protein folding, Biochem J 270 (1990) 1–16.[19] G.B. Reddy, V.R. Srinivas, N. Ahmad, A. Surolia, Molten globule-like state of

peanut lectin monomer retains its carbohydrate specificity. Implications inprotein folding and legume lectin oligomerization, J Biol Chem 274 (1999)4500–4503.

[20] R. Jaenicke, Protein folding: local structures, domains, subunits, and assemblies,Biochemistry 30 (1991) 3147–3161.

[21] K.E. Neet, D.E. Timm, Conformational stability of dimeric proteins: quantitativestudies by equilibrium denaturation, Protein Sci 3 (1994) 2167–2174.

[22] S. Batey, K.A. Scott, J. Clarke, Complex folding kinetics of a multidomain protein,Biophys J 90 (2006) 2120–2130.

[23] R. Jaenicke, R. Rudolph, in: T.E. Creighton (Ed.), Protein Structure: a PracticalApproach, IRL Press (Oxford University, Press), Oxford, 1989, pp. 191–224.

[24] N.C. Price, Assembly of multi-subunit structures, Mechanisms of protein folding,Oxford University Press, New York, 1994, pp. 160–193.

[25] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl,Current Protocols in Molecular Biology, Vo1. 1, Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., USA, 1993, pp. 16.2.1–16.2.11.

[26] J. Sambrook, E.F. Fritsch, T. Manniatis, Cold Spring Harbor Laboratories, ColdSpring Harbor, New York,; Vol. 1, 2 and 3, 2001.

[27] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head ofbacteriophage T4, Nature 227 (1970) 680–685.

[28] M.M. Bradford, A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding, Anal Biochem72 (1976) 248–254.

[29] B.J. Berger, M.H. Knodel, Characterisation of methionine adenosyltransferase fromMycobacterium smegmatis and M. tuberculosis, BMC Microbiol 3 (2003) 12.

[30] D.J. Kim, J.H. Huh, Y.Y. Yang, C.M. Kang, I.H. Lee, C.G. Hyun, S.K. Hong, J.W. Suh,Accumulation of S-adenosyl-L-methionine enhances production of actinorhodinbut inhibits sporulation in Streptomyces lividans TK23, J Bacteriol 185 (2003)592–600.

[31] A.E. Pegg, Recent advances in the biochemistry of polyamines in eukaryotes,Biochem J 234 (1986) 249–262.

[32] M.A. Grillo, S. Colombatto, S-adenosylmethionine and radical-based catalysis,Amino Acids 32 (2007) 197–202.

[33] B.A. McDaniel, F.J. Grundy, V.P. Kurlekar, J. Tomsic, T.M. Henkin, Identification of amutation in the Bacillus subtilis S-adenosylmethionine synthetase gene thatresults in derepression of S-box gene expression, J Bacteriol 188 (2006)3674–3681.

1958 V. Kamarthapu et al. / Biochimica et Biophysica Acta 1784 (2008) 1949–1958

[34] N. Hilti, R. Graub, M. Jorg, P. Arnold, A.M. Schweingruber, M.E. Schweingruber,Gene sam1 encoding adenosylmethionine synthetase: effects of its expression inthe fission yeast Schizosaccharomyces pombe, Yeast 16 (2000) 1–10.

[35] K. Ochi, E. Freese, A decrease in S-adenosylmethionine synthetase activity increasesthe probability of spontaneous sporulation, J Bacteriol 152 (1982) 400–410.

[36] M. Ferrer, T.N. Chernikova, M.M. Yakimov, P.N. Golyshin, K.N. Timmis, Chaperoninsgovern growth of Escherichia coli at low temperatures, Nat Biotechnol 21 (2003)1266–1267.

[37] M.J. Weickert, D.H. Doherty, E.A. Best, P.O. Olins, Optimization of heterologousprotein production in Escherichia coli, Curr Opin Biotechnol 7 (1996) 494–499.

[38] H.P. Sorensen, K.K. Mortensen, Soluble expression of recombinant proteins in thecytoplasm of Escherichia coli, Microb Cell Fact 4 (2005) 1.

[39] T.O. Street, D.W. Bolen, G.D. Rose, A molecular mechanism for osmolyte-inducedprotein stability, Proc Natl Acad Sci U S A 103 (2006) 13997–14002.

[40] J. Komoto, T. Yamada, Y. Takata, G.D. Markham, F. Takusagawa, Crystal structure ofthe S-adenosylmethionine synthetase ternary complex: a novel catalyticmechanism of S-adenosylmethionine synthesis from ATP and Met, Biochemistry43 (2004) 1821–1831.

[41] M.R. Eftink, R. Ionescu, Thermodynamics of protein unfolding: questions pertinentto testing the validity of the two-state model, Biophys Chem 64 (1997) 175–197.

[42] K.A. Dill, Dominant forces in protein folding, Biochemistry 29 (1990) 7133–7155.[43] P.L. Privalov, Stability of proteins: small globular proteins, Adv Protein Chem 33

(1979) 167–241.[44] M.R. Eftink, The use of fluorescence methods to monitor unfolding transitions in

proteins, Biophys J 66 (1994) 482–501.[45] A. Chatterjee, D.K. Mandal, Denaturant-induced equilibrium unfolding of

concanavalin A is expressed by a three-state mechanism and provides an estimateof its protein stability, Biochim Biophys Acta 1648 (2003) 174–183.

[46] M.S. Kumar, P.Y. Reddy, B. Sreedhar, G.B. Reddy,αB-crystallin-assisted reactivationof glucose-6-phosphate dehydrogenase upon refolding, Biochem J 391 (2005)335–341.

[47] S.M. Kelly, N.C. Price, The application of circular dichroism to studies of proteinfolding and unfolding, Biochim Biophys Acta 1338 (1997) 161–185.

[48] M.M. Sanchez del Pino, I. Perez-Mato, J.M. Sanz, J.M. Mato, F.J. Corrales, Folding ofdimeric methionine adenosyltransferase III: identification of two folding inter-mediates, J Biol Chem 277 (2002) 12061–12066.