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• W1966793804 abstract "From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5′-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations. From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5′-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations. Recent years have seen increased incidence of tuberculosis in both developing and developed countries. Information available from the complete genome sequence of Mycobacterium tuberculosis has the potential of providing the information that would generate knowledge that will enable to elucidate the unusual biology of its etiological agent, M. tuberculosis. Serine hydroxymethyltransferase (SHMT), 1The abbreviations used are: SHMT, serine hydroxymethyltransferase; PLP, pyridoxal 5′-phosphate; H4PteGlu, N 5-formyl-5,6,7,8-tetrahydropteroyl-l-glutamic acid; DTT, dithiothreitol; T m, midpoint of thermal denaturation; GdmCl, guanidinium chloride; PyP, pyridoxamine-P; FRET, fluorescence resonance energy transfer.l-serine:tetrahydrofolate 5,10-hydroxymethyltransferase is a pyridoxyl 5′-phosphate (PLP)-dependent enzyme. SHMT reaction plays a major role in cell physiology as it is considered to be a key enzyme in the pathway for interconversion of folate coenzymes that provide almost exclusively one-carbon fragments for the biosynthesis of a variety of end products such as DNA, RNA, ubiquinone, methionine, etc. (1Schirch V. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 83-112PubMed Google Scholar, 2Mc Neil J. Bognar A. Perlman R. Genetics. 1996; 142: 371-381Crossref PubMed Google Scholar, 3Shane B. Vit. Horm. 1989; 45: 263-335Crossref PubMed Scopus (288) Google Scholar). The physiological role of SHMT is the reversible interconversion of serine to glycine and irreversible hydrolysis of 5,10-CH+-H4PteGlu to 5-CHO-H4PteGlu. In addition to these physiological reactions, SHMT also catalyzes, in the absence of H4PteGlu, the retroaldol cleavage of several 3-hydroxyamino acids, such as allo-threonine, and the transamination and slow racemization of d- and l-alanine (4Shostak K. Schirch V. Biochemistry. 1988; 27: 8007-8017Crossref PubMed Scopus (49) Google Scholar, 5Contestabile, R., Paiardini, A., Pascarella, S., di Salvo, M. L., D'Aguanno, S., and Bossa, F. (2001) Eur. J. Biochem., 6508-6525Google Scholar). SHMT shows a ubiquitous distribution in nature, being found both in the prokaryotes and eukaryotes. In eukaryotes, SHMT exists as both cytosolic and mitochondrial isoforms (6Appling D. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (304) Google Scholar) encoded by the separate genes (7Garrow T. Brenner A. Whitehead M. Chen X.N. Duncan R. Korenberg J. Shane B. J. Biol. Chem. 1993; 268: 11910-11916Abstract Full Text PDF PubMed Google Scholar). A chloroplast isoform has also been reported to be present in the plants (8Besson B. Neuberger M. Rebeille F. Dounce R. Plant Physiol. Biochem. 1995; 33: 665-673Google Scholar) and Euglena gracilis (9Sakamoto M. Masuda T. Yanagimoto Y. Nakano Y. Kitaoka S. Tanigawa Y. Z. Biosci. Biotech. Biochem. 1996; 60: 1914-1944Crossref Scopus (16) Google Scholar). Three molecular forms of SHMT have been detected in choanomastigotes of Crithidia fasciculata. One of them is cytosolic, whereas the other two forms are particle bound, one being mitochondrial and the other one very likely is glycosomal (10Capelluto D.G.S. Hellnnan U. Cazzulo J.J. Cannata J.J.B. Mol. Biochem. Parasitol. 1999; 98: 187-201Crossref PubMed Scopus (12) Google Scholar). A single form of SHMT has been detected in epimastigotes of Trypanosoma cruzi (11Capelluto D.G.S. Hellnnan U. Cazzulo J.J. Cannata J.J.B. Eur. J. Biochem. 2000; 267: 712-719Crossref PubMed Scopus (21) Google Scholar). Structurally SHMT from a mammalian source is a homotetramer with a subunit molecular mass of about 53 kDa and contains 4 mol of PLP/mol of enzyme (12Renwick S.B. Snell K. Baumann U. Structure. 1998; 6: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Scarsdale J.N. Kazanina G. Radaev S. Schirch V. Biochemistry. 1999; 38: 8347-8358Crossref PubMed Scopus (73) Google Scholar). The structural details have revealed that the tetrameric enzyme is a dimer of dimers (13Scarsdale J.N. Kazanina G. Radaev S. Schirch V. Biochemistry. 1999; 38: 8347-8358Crossref PubMed Scopus (73) Google Scholar). The three SHMT isoforms found in C. fasciculata also have tetrameric structure (10Capelluto D.G.S. Hellnnan U. Cazzulo J.J. Cannata J.J.B. Mol. Biochem. Parasitol. 1999; 98: 187-201Crossref PubMed Scopus (12) Google Scholar). In contrast, the SHMT from Escherichia coli as well as from several other bacterial sources are dimeric and contain 2 mol of PLP/mol of enzyme (14Venkatesha B. Udgaonkar J.B. Rao N.A. Savithri H.S. Biochim. Biophys. Acta. 1998; 1384: 141-152Crossref PubMed Scopus (20) Google Scholar). Recently, it has been shown that the T. cruzi SHMT is present as a catalytically active monomer (11Capelluto D.G.S. Hellnnan U. Cazzulo J.J. Cannata J.J.B. Eur. J. Biochem. 2000; 267: 712-719Crossref PubMed Scopus (21) Google Scholar). X-ray crystal structure has shown that the coenzyme PLP is covalently attached to the enzyme molecule and is located at the dimer interface (12Renwick S.B. Snell K. Baumann U. Structure. 1998; 6: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Scarsdale J.N. Kazanina G. Radaev S. Schirch V. Biochemistry. 1999; 38: 8347-8358Crossref PubMed Scopus (73) Google Scholar, 14Venkatesha B. Udgaonkar J.B. Rao N.A. Savithri H.S. Biochim. Biophys. Acta. 1998; 1384: 141-152Crossref PubMed Scopus (20) Google Scholar). The folding/unfolding of both the tetrameric and the dimeric SHMTs have been extensively studied (15Cai K. Schirch D. Schirch V. J. Biol. Chem. 1995; 270: 19294-19299Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 16Cai K. Schirch V. J. Biol. Chem. 1996; 271: 2987-2994Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 17Bhatt A.N. Prakash K. Subramanya H.S. Bhakuni V. Biochemistry. 2002; 41: 12115-12123Crossref PubMed Scopus (15) Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The tetrameric SHMT undergoes a reversible unfolding with the first step being dissociation of tetramer into dimer accompanied by the removal of the enzyme-bound PLP from the active site (15Cai K. Schirch D. Schirch V. J. Biol. Chem. 1995; 270: 19294-19299Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Regarding the second step it was not clear whether the stabilized dimer of SHMT unfolds directly or does so via the formation of monomer (15Cai K. Schirch D. Schirch V. J. Biol. Chem. 1995; 270: 19294-19299Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For the dimeric SHMTs, both from E. coli and Bacillus subtilis, the first step of unfolding is dissociation of the native dimer into monomer followed by the unfolding of the stabilized monomer (16Cai K. Schirch V. J. Biol. Chem. 1996; 271: 2987-2994Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 17Bhatt A.N. Prakash K. Subramanya H.S. Bhakuni V. Biochemistry. 2002; 41: 12115-12123Crossref PubMed Scopus (15) Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). However, for the thermophilic SHMT from Bacillus stearothermophilis, a highly cooperative unfolding of the native dimer into unfolded monomer was reported (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Searches of the M. tuberculosis genome for enzymes related to SHMT have lead to the identification of two genes namely the GlyA and GlyA2, which have been proposed as putative genes encoding for SHMTs in the organism. Here we report the cloning, overexpression, and purification of the recombinant SHM1 and SHM2, the protein products of the genes GlyA and GlyA2. Furthermore, the structural, functional, and stability properties as well as the comparative studies on thermal, pH-, urea-, and GdmCl-induced unfolding of both recombinant proteins have been discussed in detail. Cloning, Overexpression, and Purification of the Proteins of M. tuberculosis GlyA and GlyA2 Genes—The complete genes of M. tuberculosis encoding functional GlyA and GlyA2 were amplified separately by PCR from the genomic DNA. The different oligonucleotides used for GlyA, based on the reported gene sequence (GenBank™ accession number Rv1093), were upstream 5′-GGAATTCCATATGTCTGCCCCGCTCGCTGAAGTT-3′ and downstream 5′-CCCAAGCTTGCGGCCGACCAGACTCCACTC-3′ for histidine-tagged protein and 5′-CCCAAGCTTTTAGCGGCCGACCAGACTCCACTC-3′ for non-histidine-tagged protein. The different oligonucleotides for GlyA2 (GenBank™ accession number Rv0070c) were upstream 5′-GGAATTCCATATGAACACCCTCAACGACTCCCTG-3′ and downstream 5′-ATAAGAATGCGGCCGCTGTACGATGCAGTTCCGGGTA-3′ for histidine-tagged protein and 5′-CCCAAGCTTTTATGTACGATGCAGTTCCGTA-3′ for non-histidine-tagged protein. Reactions were carried out in two phases for 5 and 30 cycles in a total volume of 50 μl with 2.5 units of Taq polymerase. For GlyA, the first cycle without overhang amplification was 95 °C, 1 min; 54 °C, 1 min; 72 °C, 2 min; and the second cycle with overhang amplification was 95 °C, 1 min; 60 °C, 1 min; 72 °C, 2 min with final extension for 10 min. For GlyA2, the first cycle without overhang amplification was 95 °C, 1 min; 44 °C, 1 min; 72 °C, 2 min, and the second cycle was similar to the first cycle except for the annealing temperature of 56 °C with final extension for 10 min. The amplified product was cloned into pET-22b between the NdeI and HindIII sites. DNA sequencing confirmed the homogeneity of the sequences. The resultant constructs were transformed into C41(DE3). A single colony was inoculated into 10 ml of 2× YT broth having ampicillin at a concentration of 100 μg/ml and grown overnight at 30 °C. It was then subcultured in 1 liter of 2× YT media containing similar ampicillin concentrations. For SHM1, the culture was induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside at A 600 of 0.4 to 0.5 for 12 h at 30 °C, whereas SHM2 was grown for 18 h without any induction. The cell culture was harvested followed by suspension into 50 mm potassium phosphate buffer, pH 7.8, containing 400 mm NaCl, 10 mm imidazole, 1 mm β-mercaptoethanol, and 50 μm PLP. Cells were sonicated and centrifuged to remove the cell debris. The supernatant was loaded on a nickel-nitrilotriacetic acid-agarose column pre-equilibrated with the same buffer. To remove the nonspecific binding protein constituents, the column was washed with 50 mm potassium phosphate buffer, pH 7.4, containing 400 mm NaCl and 80 mm imidazole. The protein was eluted with the same buffer containing 300 mm imidazole. The fractions containing SHMT were pooled and glycerol and DTT were added to final concentrations of 8% and 1 mm, respectively. Excess imidazole was removed by dialysis against 25 mm potassium phosphate buffer, pH 7.6, containing 50 mm NaCl, 1 mm EDTA, 8% glycerol, and 1 mm DTT. The purity of the purified proteins was checked by SDS-PAGE (19Cai K. Schirch V. J. Biol. Chem. 1996; 271: 27311-27320Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and ESI-MS and was found to be about 99% pure. Circular Dichroism Measurements—The CD measurements were made with a Jasco J810 spectropolarimeter calibrated with ammonium (+)-10-camphorsulfonate and the results were expressed as relative ellipticity and plotted as percentage values. The CD spectra were obtained at enzyme concentrations of 2 and 10 μm for far-UV CD and visible CD measurements, respectively, with a 2-mm cell at 4 °C. The values obtained were normalized by subtracting the baseline recorded for the buffer having the same concentration of denaturant under similar conditions. Synthesis of PyP-SHMT—The reduction of the PLP aldimine was achieved according to the procedure of Cai and Schirch (20Taylor R.T. Weisbach H. Anal. Biochem. 1965; 13: 80-84Crossref Scopus (168) Google Scholar). Enzyme Activity—SHMT activity of the purified recombinant proteins were determined as reported earlier (21Manohar R. Ramesh K.S. Appaji Rao N. J. Biosci. 1982; 4: 31-50Crossref Scopus (30) Google Scholar, 22Malkin L.Z. Greenberg D.M. Biochim. Biophys. Acta. 1964; 85: 117-131PubMed Google Scholar). Briefly, a 0.1-ml reaction mixture contained 400 mm potassium phosphate buffer, pH 7.6, 1.8 mm DTT, 1 mm EDTA, 50 μm PLP, 1.8 mm tetrahydrofolate, 3.6 mm l-[3-14C]serine, and approximately 1 μm protein. After incubation at 37 °C for 20 min the reaction was stopped by the addition of 100 μl of dimedone (400 mm in 50% ethanol). The reaction mixture was kept in boiling water bath for 5 min and the H-14CHO-dimedone adduct was extracted into 3 ml of toluene. A 1-ml volume of toluene extract was added to 5 ml of scintillation fluid (0.6% (w/v) 2,5-diphenyloxazole) and the radioactivity was measured on the scintillation counter. Activity was also measured by another reported method (23Schirch V. Hopkins S. Villar E. Angelaccio S. J. Bacteriology. 1985; 163: 1-7Crossref PubMed Google Scholar). A 1-ml reaction mixture contained 1–70 μg of enzyme, alcohol dehydrogenase (100 μg), NADH (250 μm), 10 mm allo-threonine in 25 mm potassium phosphate buffer, pH 7.6, 1 mm EDTA, and 1 mm DTT. The reference cuvette contained all the chemicals except l-allo-threonine. The activity was observed as decrease in the absorbance at 340 nm. The NADH consumed in the reaction was calculated using a molar extinction coefficient of 6220 m-1 cm-1. Fluorescence Measurements—Fluorescence spectra were recorded with PerkinElmer LS5B spectroluminesencemeter in a quartz cell with a 5-mm path length. All samples were incubated for 6 h under specified conditions before recording the spectra. For tryptophan fluorescence measurements the excitation wavelength was 290 nm and the spectra were recorded from 300 to 400 nm. The protein concentration used was 2 μm and the measurement was carried out at 25 °C. For FRET studies the samples were excited at 290 nm and the spectra were recorded from 300 to 450 nm. Cross-linking Using Glutaraldehyde—To the native and urea- or GdmCl-treated SHMT (1 μm enzyme), an aliquot of 25% (w/v) glutaraldehyde was added to obtain a final glutaraldehyde concentration of 1%. The samples were incubated at 25 °C for 5 min followed by quenching of the cross-linking reaction by the addition of 200 mm sodium borohydride. After incubation for 20 min, 3 μl of 10% aqueous sodium deoxycholate was added. The pH of the reaction mixtures was lowered to 2–2.5 by the addition of orthophosphoric acid, which resulted in precipitation of the cross-linked proteins. After centrifugation (13,000 × g, 4 °C, 10 min) the obtained precipitates were re-dissolved in 0.1 m potassium phosphate buffer, pH 7.6, 1% SDS, and 50 mm DTT and heated at 90–100 °C for 5 min. The molecular mass of the cross-linked products was determined by 10% SDS-PAGE (19Cai K. Schirch V. J. Biol. Chem. 1996; 271: 27311-27320Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Size Exclusion Chromatography—The gel filtration experiments were carried out on a Superdex 200HR 10/30 column (manufacturer's exclusion limit 600 kDa for proteins) on AKTA FPLC (Amersham Biosciences). The columns were equilibrated and run on 25 mm potassium phosphate buffer, pH 7.6, containing 1 mm EDTA, 100 mm NaCl, and 2 mm β-mercaptoethanol (or mentioned otherwise) and the desired urea or GdmCl concentration at 4 °C. The SHMT solution (10 μm) was incubated at the desired GdmCl or urea concentrations for 6 h at 4 °C. These samples (200 μl) were loaded on the column and run at 4 °C with a flow rate of 0.3 ml/min. pH-dependent Incubation—Recombinant SHM1 and SHM2 were dissolved in 10 mm citrate/glycine/Hepes buffer (pH of the solution maintained) of desired pH values and incubated for 4 h at 4 °C before making the measurements. Urea or Guanidinium Chloride Denaturation of SHMT—Recombinant SHM1 and SHM2 (2 μm) were dissolved in 25 mm potassium phosphate buffer, pH 7.6, containing 1 mm EDTA, 50 mm NaCl, and 1 mm DTT in the presence and absence of increasing concentrations of urea or GdmCl and incubated for 6 h at 4 °C. These samples were then used for taking various measurements. The expression of both the SHM1 and SHM2 was good and the expressed proteins were present predominantly (>90%) in the soluble fraction. The enzymes present in the soluble fraction were purified by the method described under “Experimental Procedures.” The yield of both the enzymes was in the range of 12 to 15 mg/liter. The purified proteins were homogenous as indicated by a single protein band on SDS-PAGE (Fig. 1, A and B) and a single peak in ESI-MS (data not shown). The purified enzymes were assayed for SHMT activity (21Manohar R. Ramesh K.S. Appaji Rao N. J. Biosci. 1982; 4: 31-50Crossref Scopus (30) Google Scholar, 22Malkin L.Z. Greenberg D.M. Biochim. Biophys. Acta. 1964; 85: 117-131PubMed Google Scholar) and the specific activity of 1.2 ± 0.2 and 1.8 ± 0.3 units/mg was observed for the SHM1 and SHM2, respectively. The 280/425 absorbance ratio was 4.78 and 3.43 for the purified recombinant SHM1 and SHM2, respectively, which is within the limits of the values reported for SHMT from various sources (24Andrews P. Biochem. J. 1965; 96: 569-606Crossref Scopus (2430) Google Scholar). The molecular masses of the purified recombinant SHM1 and SHM2 were determined under non-dissociating conditions as described by Andrews (25Ulevitch R.J. Kallen R.G. Biochemistry. 1997; 16: 5342-5349Crossref Scopus (78) Google Scholar) using the data of gel filtration experiments. Gel filtration of SHM1 and SHM2 on a Superdex S-200 column, calibrated with the various molecular weight standards, showed a single peak although with a slight difference in retention volume of 14.55 and 14.7 ml, respectively, for the two enzymes (Fig. 1C). When the elution volumes of the marker proteins were plotted as a function of log of molecular masses, the molecular masses of 91.2 and 87.1 kDa were obtained for SHM1 and SHM2, respectively. From the primary amino acid sequence, the molecular masses of 45.03 and 45.52 kDa were obtained for SHM1 and SHM2, respectively. The subunit masses of purified SHM1 and SHM2 were determined by SDS-PAGE. A single homogeneous protein band corresponding to molecular mass of about 45 kDa was observed for the two proteins (Fig. 1, A and B). The precise molecular weights of the two recombinant enzymes were obtained by ESI-MS experiments (data not shown). Molecular masses of 45.0 and 45.5 kDa were observed for SHM1 and SHM2, respectively, demonstrating that both the recombinant enzymes have very similar molecular masses. The results of the studies on subunit masses along with the size exclusion chromatography as reported above demonstrate that both SHM1 and SHM2 exist as dimers under physiological conditions. Furthermore, as both the proteins have similar molecular masses but significant differences are observed in their retention volumes (on size exclusion chromatography), it suggests that significant differences exist in the molecular dimensions of the two recombinant enzymes with SHM2 having a slightly more compact conformation (higher retention volume) than the SHM1 under physiological conditions. The SHMT is a PLP-dependent enzyme in which the PLP is covalently attached to the enzyme, bound as an aldimine to Lys-299 that is conserved in SHMT from various sources (12Renwick S.B. Snell K. Baumann U. Structure. 1998; 6: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Scarsdale J.N. Kazanina G. Radaev S. Schirch V. Biochemistry. 1999; 38: 8347-8358Crossref PubMed Scopus (73) Google Scholar, 14Venkatesha B. Udgaonkar J.B. Rao N.A. Savithri H.S. Biochim. Biophys. Acta. 1998; 1384: 141-152Crossref PubMed Scopus (20) Google Scholar). Visible CD studies on SHMT have shown that the visible CD signal is different between the holo- and the apo- or unfolded enzyme. The holoenzyme has a unique peak at 425 nm because of the bound PLP, which disappears on unfolding of the enzyme or on removal of PLP from the enzyme, i.e. on stabilization of apoenzyme (16Cai K. Schirch V. J. Biol. Chem. 1996; 271: 2987-2994Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The visible CD spectra of SHM1 and SHM2 are shown in Fig. 2A. For both the recombinant proteins a visible CD spectra centered at about 430 nm, showing the presence of the enzyme-bound PLP, was observed. However, at a similar enzyme concentration the visible CD signal for SHM2 was about 8 times greater than that observed for the SHM1 indicating that it probably has a higher content of PLP as compared with SHM1. To confirm this, the amount of PLP bound to the enzyme was determined for both recombinant enzymes by the method of Urevitch and Kallen (25Ulevitch R.J. Kallen R.G. Biochemistry. 1997; 16: 5342-5349Crossref Scopus (78) Google Scholar) after extensive dialysis of the purified recombinant enzymes against 25 mm potassium phosphate buffer, pH 7.6, 50 mm NaCl, and 1 mm EDTA. The SHM1 and SHM2 were found to contain 1 ± 0.2 and 2.3 ± 0.5 mol of PLP/mol of enzyme, respectively. The SHMT from mammalian sources are homotetramers and contain 4 mol of PLP/mol of enzyme (12Renwick S.B. Snell K. Baumann U. Structure. 1998; 6: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Scarsdale J.N. Kazanina G. Radaev S. Schirch V. Biochemistry. 1999; 38: 8347-8358Crossref PubMed Scopus (73) Google Scholar), whereas the SHMT from E. coli as well as from other bacterial sources are dimeric and contain 2 mol of PLP/mol of enzyme (14Venkatesha B. Udgaonkar J.B. Rao N.A. Savithri H.S. Biochim. Biophys. Acta. 1998; 1384: 141-152Crossref PubMed Scopus (20) Google Scholar). All the SHMTs reported to date including SHM2 (reported in this paper) contain 2 mol of PLP/mol of enzyme dimer, however, SHM1 was found to contain only 1 mol of PLP/mol of enzyme dimer. This is the first report on such a unique stoichiometry of PLP and enzyme for SHMT. Secondary Structure—Far-UV CD studies were carried out on both SHM1 and SHM2 to analyze the differences in the secondary structure that exist between the two enzymes. For both SHM1 and SHM2, the far-UV CD spectra characteristic of a protein having both α-helix and β-sheet secondary structure was observed (Fig. 2B). However, for similar molar concentrations of enzyme, a significantly higher ellipticity was observed for SHM2 over the whole far-UV region (Fig. 2B). This observation suggests that SHM2 have a significantly higher secondary structure as compared with SHM1. Despite having similar subunit molecular mass as SHM1, the SHM2 was found to have a more compact conformation. One possible reason for this may be the presence of a significantly higher secondary structure in SHM2, which would result in stabilization of a compact conformation. Tryptophan Fluorescence—According to the primary amino acid sequence, SHM1 has two tryptophan molecules at positions 172 and 421, whereas SHM2 has a single tryptophan at position 175. The fluorescence spectra of SHM1 and SHM2 are shown in Fig. 2C. For both the enzymes the emission wavelength maxima for the tryptophan fluorescence was observed at about 339 nm, however, there were significant differences in fluorescence intensities of the two enzymes because of the difference in number of tryptophan moieties present in them. The buried tryptophan residues in folded protein show fluorescence emission maxima at 330–335 nm, whereas on unfolding of protein the tryptophan fluorescence emission maxima shifts to about 350 nm (27Angelaccio S. Pascarella S. Fattori E. Bossa F. Strong W. Schirch V. Biochemistry. 1992; 31: 155-162Crossref PubMed Scopus (31) Google Scholar). Hence, both in SHM1 and SHM2 the tryptophan molecule(s) is not completely buried but partially exposed to the solvent. Fluorescence Resonance Energy Transfer—For eSHMT it has been demonstrated that reduction of the PLP aldimine bond results in absorbance maxima at 355 nm that overlaps the emission spectrum of tryptophan. Hence, the PyP can be used as an energy acceptor of tryptophan fluorescence. For native eSHMT FRET is observed that abolishes on unfolding of enzyme (20Taylor R.T. Weisbach H. Anal. Biochem. 1965; 13: 80-84Crossref Scopus (168) Google Scholar). Fig. 2, D and E, summarizes the results of FRET experiments on SHM1 and SHM2. For both the SHM1 and SHM2 in the native conformation two emission maxima, one around 339 nm (tryptophan fluorescence) and the other at 385 nm (PyP emission resulting from FRET) were observed. However, on unfolding of enzymes by incubation in 8 m urea only single emission maximum at 352 nm was observed. These observations demonstrate that for SHM1 and SHM2 in the native conformation there is FRET between tryptophan residues and the bound PLP suggesting that the tryptophan residue(s) is located less than 5 Å from PyP in the native conformation of these two enzymes and on denaturation of the enzymes these two moieties move apart resulting in the loss of FRET. Functional Properties—All the forms of SHMT for which a primary sequence is known contain the eight-residue conserved sequence, VTTTTHK(Pyr)T, near the active-site lysyl residue (Lys-229 as in eSHMT) that forms the internal aldimine with pyridoxal phosphate. The active site octa-peptide from SHMT is unusual in the sense that it has five threonine residues conserved in all the reported primary sequence of SHMTs (Fig. 3). Mutation studies have demonstrated that Thr-226 plays an important role in converting the gem-diamine complex to external aldimine complex. A T226A mutant of eSHMT has been shown to distinguish between substrates and substrate analogs in formation of the gem-diamine complex (28Schirch L. Jenkins N.J. J. Biol. Chem. 1964; 238: 3797-3800Abstract Full Text PDF Google Scholar). The primary sequence of SHM1 and SHM2 show significant changes in the conserved threonine sequence (Fig. 3). In SHM1, the Thr-225 (corresponding to Thr-227 of eSHMT) and Thr-222 (corresponding to Thr-224 of eSHMT) are replaced with valine and serine, respectively. In SHM2 the Thr-226 (corresponding to Thr-225 of eSHMT) is replaced by serine (Fig. 3). These alterations may lead to significant changes in substrate binding and complex formation in these enzymes. It has been reported that d-alanine reacts with SHMT to undergo a slow half-transamination reaction resulting in the formation of apoenzyme, pyridoxamine-P, and pyruvate (29Schirch V. Mason M. J. Biol. Chem. 1962; 237: 2578-2581Abstract Full Text PDF Google Scholar). Experimentally this reaction is detected by a decrease of the absorption" @default.
• W1966793804 created "2016-06-24" @default.
• W1966793804 creator A5082723762 @default.
• W1966793804 creator A5083338567 @default.
• W1966793804 date "2003-10-01" @default.
• W1966793804 modified "2023-10-03" @default.
• W1966793804 title "Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis" @default.
• W1966793804 cites W115375044 @default.
• W1966793804 cites W1563081877 @default.
• W1966793804 cites W1586384817 @default.
• W1966793804 cites W1599498003 @default.
• W1966793804 cites W1708740028 @default.
• W1966793804 cites W1788126413 @default.
• W1966793804 cites W1833006673 @default.
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