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• W2078691421 abstract "The methyltetrahydrofolate (CH3-H4folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH3-H4folate to cob(I)amide. This key step in anaerobic CO and CO2 fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH3-H4folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH3-H4folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH3-H4folate binding. An N199A variant exhibits only ∼20-fold weakened affinity for CH3-H4folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer. The methyltetrahydrofolate (CH3-H4folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH3-H4folate to cob(I)amide. This key step in anaerobic CO and CO2 fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH3-H4folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH3-H4folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH3-H4folate binding. An N199A variant exhibits only ∼20-fold weakened affinity for CH3-H4folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer. The methyltetrahydrofolate (CH3-H4folate) 5The abbreviations used are: CH3-H4folate, methyltetrafolate; MetH, cobalamin-dependent methionine synthase; MeTr, CH3-H4folate:corrinoid iron-sulfur protein methyltransferase; MtrH, methyltetrahydromethanopterin: cob(I)alamin methyltransferase; DHFR, dihydrofolate reductase; PNP, purine nucleoside phosphorylase; DHPS, dihydropteroate synthases; ImmH, immucillin-H; PDB, Protein Data Bank; CFeSP, corrinoid-iron-sulfur protein; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; ITC, isothermal titration calorimetry; r.m.s.d., root mean square deviation. corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) from Moorella thermoacetia catalyzes the methyl group transfer from CH3-H4folate to cob(I)amide (Reaction 1) in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation (1Ragsdale S.W. Horvath I.T. Iglesia E. Klein M.T. Lercher J.A. Russell A.J. Stiefel E.I. Encyclopedia of Catalysis. John Wiley and Sons, Inc., New York2003: 452-467Google Scholar, 2Ragsdale S.W. CRC Crit. Rev. Biochem. Mol. Biol. 2004; 39: 165-195Crossref PubMed Scopus (303) Google Scholar) to generate an organometallic methyl-Co species on the CFeSP. The methylated CFeSP then reacts with carbon monoxide and CoA to generate acetyl-CoA in a reaction catalyzed by the bifunctional NiFeS protein, carbon monoxide dehydrogenase/acetyl-CoA synthase (2Ragsdale S.W. CRC Crit. Rev. Biochem. Mol. Biol. 2004; 39: 165-195Crossref PubMed Scopus (303) Google Scholar). The MeTr reaction is similar to a variety of cobalamin-dependent methylation reactions, including methyl transfer from CH3-H4folate to the bound cobalamin cofactor of methionine synthase, which subsequently transfers its methyl group from methylcobalamin to homocysteine (3Banerjee R. Ragsdale S.W. Ann. Rev. Biochem. 2003; 72: 209-247Crossref PubMed Scopus (593) Google Scholar, 4Matthews R.G. Acc. Chem. Res. 2001; 34: 681-689Crossref PubMed Scopus (224) Google Scholar). CH3−CH4folate+Co(I)−CFeSP→CH3−Co(III)−CFeSP+H4folate REACTION 1 The MeTr reaction mechanism consists of several steps (5Seravalli J. Zhao S. Ragsdale S.W. Biochemistry. 1999; 38: 5728-5735Crossref PubMed Scopus (34) Google Scholar). The first two steps include a pH-dependent conformational change followed by binding of CH3-H4folate. Equilibrium dialysis studies shows that MeTr has a high affinity binding site for CH3-H4folate with a Kd of 10 μm (6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar). CH3-H4folate binds to MeTr in the unprotonated state and then undergoes rapid protonation (5Seravalli J. Zhao S. Ragsdale S.W. Biochemistry. 1999; 38: 5728-5735Crossref PubMed Scopus (34) Google Scholar, 6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar). This proton transfer step generates a positive charge at the N5 position of the pterin ring, resulting in electrophilic activation of the methyl group of CH3-H4folate (3Banerjee R. Ragsdale S.W. Ann. Rev. Biochem. 2003; 72: 209-247Crossref PubMed Scopus (593) Google Scholar). This activation step is important because H4folate is a very poor leaving group, but it is not rate-limiting. Next, the CFeSP binds, and its Co(I) center catalyzes an SN2 displacement of the N5 methyl group of CH3-H4folate to form the methylated CFeSP. There is evidence that proton transfer to the pterin occurs in a tertiary complex with CH3-H4folate and cob(I)alamin for the cobalamin-dependent methionine synthase (MetH) (7Smith A.E. Matthews R.G. Biochemistry. 2000; 39: 13880-13890Crossref PubMed Scopus (32) Google Scholar) and in the ternary complex with CH3-H4folate and homocysteine for the enzyme cobalamin-independent methionine synthase (8Taurog R.E. Matthews R.G. Biochemistry. 2006; 45: 5092-5102Crossref PubMed Scopus (10) Google Scholar). Interestingly, the crystal structure of MeTr (in the absence of substrate) reveals no obvious proton donor within H-bonding distance of the N5 position of CH3-H4folate, which was modeled into the structure (9Doukov T. Seravalli J. Stezowski J. Ragsdale S.W. Structure (Camb.). 2000; 8: 817-830Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The only amino acid that located near enough to N5 to participate in H-bonding is the side chain of Asn-199; however, this group is not chemically suitable to act as a proton donor. Thus, the mechanism of proton transfer leading to electrophilic activation of the methyl group is unclear. To solve this paradox, one might posit that the proton donor moves into place after CH3-H4folate binds; however, the recent crystal structure of the CH3-H4folate-MetH complex shows that there is no acidic group positioned near the N5 group of the pterin and, like the MeTr structure, Asn-199 (MeTr numbering) is within H-bonding distance to N5 (10Evans J.C. Huddler D.P. Hilgers M.T. Romanchuk G. Matthews R.G. Ludwig M.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3729-3736Crossref PubMed Scopus (116) Google Scholar) (Fig. 1). The lack of discernible proton delivery systems in the crystal structures of enzymes that catalyze proton transfer is surprisingly common. Besides methyltransferases, this situation is encountered in a number of enzymes, including dihydrofolate reductase (DHFR) (11Rod T.H. Brooks III, C.L. J. Am. Chem. Soc. 2003; 125: 8718-8719Crossref PubMed Scopus (43) Google Scholar) and purine nucleoside phosphorylase (PNP) (12Fedorov A. Shi W. Kicska G. Fedorov E. Tyler P.C. Furneaux R.H. Hanson J.C. Gainsford G.J. Larese J.Z. Schramm V.L. Almo S.C. Biochemistry. 2001; 40: 853-860Crossref PubMed Scopus (202) Google Scholar). The latter catalyzes the reversible phosphorolysis of purine nucleoside to form the corresponding purine base and ribose 1-phosphate. Fig. 1 compares the H-bonding patterns in MeTr, MetH, and DHFR near the N5 of CH3-H4folate (or folate in DHFR) with PNP in the vicinity of N7 of the transition state analog immucillin-H (ImmH). In addition to the structural homology, there are mechanistic similarities of the methyltransferases with PNP as well. To facilitate their bond cleavage reactions, both sets of enzymes appear to require substrate protonation and subsequent stabilization of a protonated species in the transition state. Evidence that N7 is protonated in the transition state of the PNP reaction comes from a series of elegant studies by Schramm and co-workers using transition state analogs (13Lewandowicz A. Ringia E.A. Ting L.M. Kim K. Tyler P.C. Evans G.B. Zubkova O.V. Mee S. Painter G.F. Lenz D.H. Furneaux R.H. Schramm V.L. J. Biol. Chem. 2005; 280: 30320-30328Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 14Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Shi W. Fedorov A. Lewandowicz A. Cahill S.M. Almo S.C. Schramm V.L. Biochemistry. 2002; 41: 14489-14498Crossref PubMed Scopus (64) Google Scholar). They find a difference in affinity of 107 (10.1 kcal/mol) between ImmH, an analog that is an H-bond donor at N7 (always protonated) and 4-aza-3-deaza-ImmH, an analog that is an H-bond acceptor at N7 (never protonated), suggesting that analogs that are protonated at N7 mimic the transition state. Available to accept the H-bond from N7 of substrate is Asn-243 (12Fedorov A. Shi W. Kicska G. Fedorov E. Tyler P.C. Furneaux R.H. Hanson J.C. Gainsford G.J. Larese J.Z. Schramm V.L. Almo S.C. Biochemistry. 2001; 40: 853-860Crossref PubMed Scopus (202) Google Scholar, 13Lewandowicz A. Ringia E.A. Ting L.M. Kim K. Tyler P.C. Evans G.B. Zubkova O.V. Mee S. Painter G.F. Lenz D.H. Furneaux R.H. Schramm V.L. J. Biol. Chem. 2005; 280: 30320-30328Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The oxygen of the carboxamide side chain of Asn-243 is 3.3 Ä away from N7 with an unprotonated substrate bound and moves closer (2.8-2.9 Ä) when protonated substrate analogs are bound (12Fedorov A. Shi W. Kicska G. Fedorov E. Tyler P.C. Furneaux R.H. Hanson J.C. Gainsford G.J. Larese J.Z. Schramm V.L. Almo S.C. Biochemistry. 2001; 40: 853-860Crossref PubMed Scopus (202) Google Scholar, 13Lewandowicz A. Ringia E.A. Ting L.M. Kim K. Tyler P.C. Evans G.B. Zubkova O.V. Mee S. Painter G.F. Lenz D.H. Furneaux R.H. Schramm V.L. J. Biol. Chem. 2005; 280: 30320-30328Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). NMR studies show that a single H-bond between Asn-243 and N7 of substrate is only partly responsible for the full 10.1 kcal/mol difference between ImmH and 4-aza-3-deaza-ImmH binding affinity (13Lewandowicz A. Ringia E.A. Ting L.M. Kim K. Tyler P.C. Evans G.B. Zubkova O.V. Mee S. Painter G.F. Lenz D.H. Furneaux R.H. Schramm V.L. J. Biol. Chem. 2005; 280: 30320-30328Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 14Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Shi W. Fedorov A. Lewandowicz A. Cahill S.M. Almo S.C. Schramm V.L. Biochemistry. 2002; 41: 14489-14498Crossref PubMed Scopus (64) Google Scholar); however, this bond appears to be enmeshed within an extended H-bonding network (Fig. 1) that is responsible for this transition state stabilization. The equivalent residue to Asn-243 in MeTr, Asn-199, is conserved in all methyltransferases. Given the provocative location of Asn-199 in MeTr and its potential role in transition state stabilization in the transmethylation reaction, we performed site-directed mutagenesis, kinetic, and structural studies to evaluate the contribution of this residue to catalysis. General DNA Manipulations—DNA isolation and manipulation were performed using standard techniques (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual.2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmid DNA was purified with a QIAprep spin miniprep kit (Qiagen, Valencia, CA). DNA fragments were purified from agarose gels using a QIAquick gel extraction kit (Qiagen). The DNA sequences of all PCR-generated DNA fragments were confirmed by automated sequencing of both strands by the Genomics Core Research Facility (University of Nebraska-Lincoln, NE) with a Beckman Coulter CEQ2000XL 8-capillary DNA sequencer using dye terminator chemistry. Site-directed Mutagenesis of MeTr—Asn-199 was mutated to Ala using the QuikChange site-directed mutagenesis protocol from Stratagene (La Jolla, CA) using the wild-type pET3a-MeTr plasmid (9Doukov T. Seravalli J. Stezowski J. Ragsdale S.W. Structure (Camb.). 2000; 8: 817-830Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) as a template. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 (Invitrogen) cells. The N199A mutant plasmid was then isolated and transformed into B834(DE3)pLysS(met-) (Novagen) E. coli cells. Enzyme Purification—E. coli cells transformed with pET3a-MeTr (N199A mutant or wild-type) were grown in 3 or 4 liters of Terrific Broth essentially as described earlier (9Doukov T. Seravalli J. Stezowski J. Ragsdale S.W. Structure (Camb.). 2000; 8: 817-830Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The cells were induced with 1 mm isopropylthio-β-d-galactoside when the OD at 600 nm reached 0.5-0.8, grown for 3 h at 37 °C or 11 h at 25 °C, and then harvested by centrifugation at 5,000 rpm for 30 min. The cells (12 or 16 g) were suspended in 50 or 40 ml of sonication buffer (50 mm Tris, pH 7.6, containing 2 mm 1,4-dithio-dl-threitol (DTT), 1 mg/ml lysozyme, 1 units/ml DNase I, and 0.1 mg/ml phenylmethylsulfonyl fluoride) and disrupted by sonication for 10 min at 0 °C. After centrifugation at 32,000 rpm for 90 min at 4 °C with a Beckman Ti35 rotor, the supernatant fraction was recovered as cell-free extract. The cell-free extract was heated at 70 °C for 40 min and then centrifuged at 10,000 rpm for 15 min at 4 °C. MeTr was then purified from the cell-free extract using a 150-ml phenyl-Sepharose column and buffer-exchanged and concentrated by ultrafiltration into 50 mm Tris, pH 7.6, containing 0.1 m NaCl and 2 mm DTT. The concentration of protein was determined with the rose bengal method (16Elliott J.I. Brewer J.M. Arch. Biochem. Biophys. 1978; 190: 351-357Crossref PubMed Scopus (90) Google Scholar). A typical yield from one liter of culture was 50 mg of MeTr. Activity Measurements—For steady-state assays, MeTr activity was determined in the reverse direction by following transfer of the methyl group of methylcobalamin to H4folate as described earlier (17Zhao S.Y. Ragsdale S.W. Biochemistry. 1996; 35: 2476-2481Crossref PubMed Scopus (13) Google Scholar). Briefly, the assay was performed at pH 8.4 and 55 °C using 66 μm methylcobalamin, 300 μm H4folate, and the decrease in absorbance at 520 nm from methyl-Co(III) was measured. The forward reaction was measured essentially as described earlier (17Zhao S.Y. Ragsdale S.W. Biochemistry. 1996; 35: 2476-2481Crossref PubMed Scopus (13) Google Scholar) at 25 °C in a reaction mixture containing 0.1 m potassium succinate, pH 5.0, 60 mm NaCl, 3.6 μm CFeSP, 87 μm CH3-H4folate, MeTr (4.1 nm monomer for the wild-type or 8.3 μm monomer for the N199A variant) and 165 μm Ti(III)-citrate to reduce Co(II) to the Co(I) state. The decrease in absorbance at 390 nm (Co(I) decay) and increase at 450 (methyl-Co(III) formation) were measured. The absorbance changes for the forward and reverse reactions were monitored using an OLIS Cary-14-converted spectrometer (Bogart, GA). Presteady-state kinetic experiments for the wild-type MeTr and N199A variant were performed at 25 °C under anaerobic conditions. For both experiments, 300 μm 6S-CH3-H4folate, MeTr (40 μm of monomer), 1 mm Ti(III)citrate, 0.1 m MES, pH 5.1, 0.1 m NaCl was rapidly mixed with 10 μm CFeSP, 1 mm Ti(III)citrate (to reduce the CFeSP to the Co(I) state), 0.1 m MES, pH 5.1, 0.1 m NaCl. The decrease in absorption of Co(I) at 390 and the increase in absorption of the methylated Co(III) at 450 nm were recorded. For the CFeSP, the Δε between cob(I)amide and methylcob(III)amide at 390 and 450 nm is 17 and 6 mm-1 cm-1, respectively (18Zhao S. Roberts D.L. Ragsdale S.W. Biochemistry. 1995; 34: 15075-15083Crossref PubMed Scopus (48) Google Scholar). The experiment with the wild-type protein was performed with a DX.17MV sequential stopped-flow ASVD spectrophotometer from Applied Photophysics (Leatherbarrow, England), whereas for the N199A variant MeTr, the same experiments were performed at room temperature using an S2000 miniature fiber optic spectrometer (Ocean Optics, Inc., Dunedin, FL) inside a Vacuum Atmospheres (Hawthorne, CA) anaerobic chamber. Binding Studies—Equilibrium dialysis experiments were performed as described (6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar) using a lower solution containing 40 μm N199A-MeTr monomer and an upper solution containing varying concentrations of (6R,S)-14CH3-H4folate (4-75 μm). Both solutions contained 0.1 mm MES, pH 6.1, and 1 mm DTT. Fluorescence quenching experiments were performed at pH 7.6 by titrating 2.8 μm MeTr monomer with CH3-H4folate as described (6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar). We could not measure the Kd at low pH values because under these conditions, the fluorescence of CH3-H4folate was much more intense than that of MeTr. Isothermal titration calorimetry (ITC) experiments for wild-type and N199A MeTr were performed with a MicroCal VPITC isothermal titration calorimeter (MicroCal Inc., Northampton, MA) at pH 5.1 and 7.6. Before the experiment, the enzyme solutions were dialyzed against the buffer with appropriate pH, i.e. 0.1 m MES, pH 5.1, or 50 mm Tris, pH 7.6, containing 0.1 m NaCl and 2 mm DTT if needed for anaerobiosis. The reaction cell was filled with a 0.05 mm (as monomer) MeTr solution. The titrated ligand [6S]-5-CH3-H4folate (1.5 mm) was dissolved in the same buffer used for dialysis of the enzyme. The stirring speed was 310 rpm, and the thermal power of 29 injections of 10 μl was recorded every 120 s. Thermogram analysis was performed using the Origin 7.0 software supplied with the instrument. Crystal Growth—Crystals of MeTr with CH3-H4folate (CH3-H4folate/MeTr) and of N199A with CH3-H4folate (CH3-H4folate/N199A) were produced at room temperature using a hanging drop setup and equal amounts of protein and precipitant (typically 2 μl of protein to 2 μl of precipitant). The protein solutions were kept in an anaerobic bottle before crystallization. Small aliquots were taken by syringe for the experiments. Typical protein solution contained 15-25 mg/ml MeTr in 50 mm Tris pH 7.6, 100 mm NaCl, 2 mm DTT. For the complexes with CH3-H4folate, a 3-fold molar excess of the 6-S-monoglutarate substrate (Schricks Laboratories, Jona, Switzerland) was added to the crystallization mixture. A precipitant stock solution contained 8-15% (w/v) polyethylene glycol monomethyl ether 5000, 20-50 mm calcium acetate, 50 mm HEPES, pH 7.5, and 20% glycerol. To obtain single crystals, this stock solution was diluted 50-100-fold in 20% glycerol prior to crystallization. Typically, 4 μl of the stock mixture were added to either 196 μl or 396 μl of 20% glycerol to make the working crystallization solution. Crystals formed two-dimensional plates and were very fragile and difficult to manipulate. All crystals were cryofrozen directly from the drops into the cryostream. The 20% glycerol in the crystallization condition acted as cryoprotectant. Data Collection—The CH3-H4folate/MeTr data set was obtained at the National Synchrotron Light Source (NSLS, Brookhaven National Laboratory) X12-C line on a B4 Brandeis CCD 4 cell detector. The CH3-H4folate/N199A data set was also collected at NSLS on the X25 beam line with a MAR345 image plate detector. Diffraction data were integrated with MOSFLM (19Leslie A.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 48-57Crossref PubMed Scopus (972) Google Scholar) and scaled and merged using SCALA (20Evans P.R. Protein Crystallogr. 1997; 33: 22-24Google Scholar) and TRUNCATE (21French G.S. Wilson K.S. Acta Crystallogr. Sect. A. 1978; 34: 517-534Crossref Scopus (895) Google Scholar) programs in the CCP4 suite (22Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Data statistics are shown in Table 1.TABLE 1Data collection and refinement statisticsWild-type with CH3-H4folateN199 A with CH3-H4folateDataWavelength (Ä)1.30000.91938Space groupP212121P212121Unit cell dimensions (Ä)50.16 78.55 135.6250.08 78.17 135.98Resolution (Ä)30.92-2.20 (2.26-2.20)aValues in parentheses are for the highest resolution bin46.73-2.30 (2.36-2.30)Reflections total/unique103105/26806 (6074/1750)130051/24151 (8478/1752)Completeness96.2 (87.9)99.1 (99.3)Multiplicity3.8 (3.5)5.4 (4.8)RsymbRsym = ∑|Ii - Im|/∑Ii where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry related reflections. (%)0.086 (0.206)0.134 (0.385)Mean (I)/sd(I)11.5 (5.3)11.1 (4.3)Wilson B factor (Ä2)14.817.2RefinementResolution (Ä)30.88-2.20 (2.26-2.20)40.19-2.30 (2.36-2.30)Rcryst/RfreecRcryst, Rfree = ∑||Fo| - |Fc||/∑|Fo where the working (cryst) and the free R-factors are calculated using the working and the test (free) reflection sets, respectively. The test reflections were held aside throughout the refinement. (%)16.26/22.40 (17.4/25.0)16.77/22.37 (17.40/24.11)r.m.s.d. bonds (Ä)0.0070.007r.m.s.d. angle (degree)1.0491.078Reflections in working/test set24177/2586 (1571/165)21756/2369 (1588/158)Nonhydrogen protein atoms44854358Nonhydrogen heteroatoms2 Ca + 64 in CH3-H4folate2 Ca + 64 in CH3-H4folateWaters426357Average B-factor, all atoms (Ä2)9.3811.36CH3-H4folate13.419.5Correlation coefficientdFo - Fc/Fo - Fcfree.0.947/0.9030.946/0.905a Values in parentheses are for the highest resolution binb Rsym = ∑|Ii - Im|/∑Ii where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry related reflections.c Rcryst, Rfree = ∑||Fo| - |Fc||/∑|Fo where the working (cryst) and the free R-factors are calculated using the working and the test (free) reflection sets, respectively. The test reflections were held aside throughout the refinement.d Fo - Fc/Fo - Fcfree. Open table in a new tab Structure Determination—The crystal structure of CH3-H4folate/MeTr was solved by molecular replacement using the structure of MeTr in space group P212121 (PDB code 1F6Y) as the search model. In particular, a MeTr dimer model with all protein atoms and no water molecules was used for the initial molecular replacement in the program EPMR (23Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (691) Google Scholar), yielding a correct solution with a correlation coefficient CC = 0.538 and a residual factor r = 0.466. Unaccounted for electron density in the TIM barrel was modeled as CH3-H4folate with restraints generated from the HIC-Up server. Electron density fitting was performed initially in XtalView (24McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) and later in COOT (25Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). The structure was refined with TLS restrained refinement parameters without a σ cutoff and with loose NCS restraints as implemented in REFMAC (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar, 27Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1654) Google Scholar). Since the publication of the apo-MeTr structure, a sequence revision was published for the residues 224-225 (Met-224-Ser-225 to Asp-224-Ala-225). These changes have been incorporated into the current structures. The model also contains a cis-peptide bond between residues Asn-96 and Ser-97 in both subunits and another one between Asn-188 and Pro-189 in the ordered loop of subunit A. In addition, a 6.5-σ electron density peak suggests the presence of a metal ion near residue Asp-224. A metal ion modeled into this electron density is in position to interact with Asp-224, five water molecules, and the carboxyl group of Gly-222, with distances ranging from 2.38 to 2.75 Ä. Based on these oxygen-rich interactions, the bond lengths, and the σ of the electron density peak, a divalent cation such as calcium (present in the crystallization conditions) is a reasonable assignment. In subunit A, the putative calcium ion is well ordered, but in subunit B, it has much weaker density and was modeled at 50% occupancy. The final model for CH3-H4folate/MeTr has excellent stereochemistry (Table 1), and no residues in the disallowed region of the Ramachandran plot (440 of 469 non-glycine and non-proline residues were in the most favored region, 29 were in the additional allowed region, 0 were in the generously allowed region, and 0 were in the disallowed region). The loop after the sixth helix (184-188) in subunit B is disordered, and these residues were omitted from the final model. The final model has 519 protein residues of MeTr, two calcium atoms, two CH3-H4folate molecules, and 426 ordered water molecules. The CH3-H4folate/N199A structure was solved to 2.3 Ä resolution using rigid body refinement and TLS restrained refinement in REFMAC starting from the CH3-H4folate/MeTr model and keeping analogous reflections in the test set for the Rfree reference. The model was modified by changing Asn-199 to alanine. A Ramachandran plot shows that 440 of 469 non-glycine and non-proline residues are in the most favored region, 28 are in additional allowed region, 1 is in generously allowed region (leucine 162, subunit A), and 0 is in the disallowed region. The final model has 519 protein residues of MeTr, two calcium atoms, two CH3-H4folate molecules, and 357 ordered water molecules. As in the CH3-H4folate/MeTr model, the loop after the sixth helix (184-188) in subunit B is disordered, and these residues were omitted from the final model. Composite omit maps calculated in CNS (28Brunger A.T. J. Mol. Biol. 1988; 203: 803-816Crossref PubMed Scopus (361) Google Scholar) were used to check both the CH3-H4-folate/MeTr and the CH3-H4-folate/N199A structures. Effect of Asn-199 on CH3-H4 folate Binding—Since Asn-199 is in hydrogen-bonding contact with N5 of CH3-H4folate, we expected that the N199A mutation would decrease affinity of MeTr for this substrate. However, we do not observe a large decrease in affinity. Based on equilibrium dialysis experiments with the N199A variant and 6S-CH3-H4folate (Fig. 2A), the Kd value is 26.5 ± 8.8 μm, and the enzyme reaches a maximum ratio of 0.95 ± 0.13 mol of CH3-H4folate bound per mol of MeTr at pH 6.1. With the wild-type protein, the Kd value was determined by equilibrium dialysis to be pH-independent between pH 4.9 and 8.5 and is about 2.6-fold lower (10 ± 1 μm) (6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar). Tryptophan residue(s) undergo fluorescence quenching when MeTr binds CH3-H4folate (Fig. 2B) (6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar, 17Zhao S.Y. Ragsdale S.W. Biochemistry. 1996; 35: 2476-2481Crossref PubMed Scopus (13) Google Scholar). The Kd value obtained from tryptophan fluorescence quenching experiments is 21.3 ± 1.0 μm for the N199A variant at pH 7.6, which compares with a value between 0.64 μm (17Zhao S.Y. Ragsdale S.W. Biochemistry. 1996; 35: 2476-2481Crossref PubMed Scopus (13) Google Scholar) and 2.1 μm (6Seravalli J. Shoemaker R.K. Sudbeck M.J. Ragsdale S.W. Biochemistry. 1999; 38: 5736-5745Crossref PubMed Scopus (21) Google Scholar), which were measured earlier for the wild-type protein at pH 7.6. ITC experiments were performed for" @default.
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