Catalytic and Thermodynamic Properties of Tetrahydromethanopterin-dependent Serine Hydroxymethyltransferase from Methanococcus jannaschii

Abstract

The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of Cβ of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C1) units for several essential biosynthetic processes. In many Archaea, C1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability. The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of Cβ of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C1) units for several essential biosynthetic processes. In many Archaea, C1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability. Serine hydroxymethyltransferase (SHMT, 1The abbreviations used are: SHMT, serine hydroxymethyltransferase; mjSHMT, M. jannaschii SHMT; eSHMT, E. coli SHMT; H4MPT, tetrahydromethanopterin; H4PteGlu, tetrahydropteroylglutamate or tetrahydrofolate; DTT, dithiothreitol; PLP, pyridoxal phosphate; SVD, singular value decomposition algorithm. EC 2.1.2.1) catalyzes the reversible transfer of Cβ of serine to tetrahydropteroylglutamate (H4PteGlu) to form glycine and 5,10-methylene-H4PteGlu. In Eukarya and Eubacteria, H4PteGlu functions as a carrier of C1 units in several oxidation states, which are used in the biosynthesis of important cellular components, such as purines and thymidylate, in the regeneration of methionine from homocysteine or, in acetogenic bacteria, in the synthesis of acetyl-CoA. The reaction catalyzed by SHMT represents in these organisms one of the major loading routes of C1 units onto the folate carrier (1Schirch L. Blakley R.L. Benkovic S.J. Folates and Pterins. Vol I. John Wiley & Sons, Inc., New York1984: 399-431Google Scholar). In methanogens and several other Archaea, C1 fragments from formyl to methyl oxidation levels are carried by tetrahydromethanopterin (H4MPT), a pterin-containing compound involved in methanogenesis. Although H4PteGlu and H4MPT are structurally similar in their pterin-like portion (Fig. 1) and in the role as C1 units carriers, they are functionally distinct. H4MPT does not appear to be suited to most of the biosynthetic functions of H4PteGlu. Moreover, the biosynthetic pathways of the two carriers have few, if any, homologies, suggesting the possibility of separate evolutionary origins (2Maden B.E. Biochem. J. 2000; 350: 509-529Crossref Google Scholar). In the metabolism of folates, SHMT represents a unique link between Archaea and the rest of living beings, in the sense that, whereas all SHMTs clearly share a common evolutionary origin (3Paiardini A. Gianese G. Bossa F. Pascarella S. Proteins. 2003; 50: 122-134Crossref PubMed Scopus (30) Google Scholar), other enzymes that use H4MPT as cofactor do not show any significant homology to their eukaryotic and eubacterial counterparts (2Maden B.E. Biochem. J. 2000; 350: 509-529Crossref Google Scholar). Although a gene encoding SHMT is present in all archaeal genomes so far sequenced, little information is available on the catalytic properties and metabolic role of the enzyme in these organisms. Modified folates are not commercially available, and this has clearly hindered a satisfactory characterization of archaeal SHMTs. Moreover, the purification of the enzyme from Archaea that thrive in extreme environments is complicated by the difficulty of growing these organisms in a laboratory. Two reports of purified SHMT activity, from Methanobacterium thermoautotrophicum (4Hoyt J.C. Oren A. Escalante-Semerena J.C. Wolfe R.S. Arch. Microbiol. 1986; 145: 153-158Crossref Scopus (22) Google Scholar), recently renamed Methanothermobacter marburgensis (5Wasserfallen A. Nolling J. Pfister P. Reeve J. Conway de Macario E. Int. J. Syst. Evol. Microbiol. 2000; 50: 43-53Crossref PubMed Scopus (179) Google Scholar), and from Sulfolobus solfataricus (6Delle Fratte S. White R.H. Maras B. Bossa F. Schisch V. J. Bacteriol. 1997; 179: 7456-7461Crossref PubMed Google Scholar), with limited structural and functional characterization, have been made. In the first report, the enzyme was proposed to function in vivo in the direction of serine biosynthesis. Importantly, both works provided evidence that SHMT was selective toward the modified folate used by the source organisms: H4MPT for M. marburgensis and sulfopterin for S. solfataricus (2Maden B.E. Biochem. J. 2000; 350: 509-529Crossref Google Scholar, 7White R.H. J. Bacteriol. 1991; 173: 1987-1991Crossref PubMed Google Scholar). This study reports on the purification of recombinant SHMT from Methanococcus jannaschii (mjSHMT) and the characterization of its catalytic and thermodynamic properties. The aim of the research was to assess the extent to which mjSHMT is structurally and mechanistically similar to its prokaryotic and eukaryotic counterparts. Materials—Ingredients for bacterial growth were from Difco. Chemicals used in the purification procedure were from BDH, and DEAE-Sepharose and phenyl-Sepharose were from Amersham Biosciences. Enzymes used for the cloning procedures were purchased from Roche Diagnostics. SHMT from Escherichia coli (eSHMT) was expressed and purified as described previously (8Schirch V. Hopkins S. Villar E. Angelaccio S. J. Bacteriol. 1985; 163: 1-7Crossref PubMed Google Scholar). The oligonucleotides used in the PCR reaction were from MWG Biotech. (6S)-H4PteGlu was a gift from A. G. Eprova, Schaffhausen, Switzerland. All other reagents were from Sigma-Aldrich. The bifunctional enzyme methylene-H4PteGlu dehydrogenase/methenyl-H4PteGlu cyclohydrolase from E. coli was purified from a recombinant E. coli bacterial strain kindly provided by Dr. Martino di Salvo (Università “La Sapienza,” Rome, Italy) following the procedure described previously (9Fu T.F. di Salvo M. Schirch V. Anal. Biochem. 2001; 290: 359-365Crossref PubMed Scopus (16) Google Scholar). NADP-dependent methylene-H4MPT dehydrogenase from Methylobacterium extorquens AM1 and H4MPT purified from M. marburgensis were kindly provided by Rolf Thauer (Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany). The thermostable alcohol dehydrogenase from Thermoanaerobium brockii employed in the threonine aldolase assay was from Sigma-Aldrich. Cloning of the glyA Gene from M. jannaschii—A pUC18 plasmid with a 1686-bp insert containing the gene encoding mjSHMT was purchased from the American Type Culture Collection. This plasmid (AMJK15) was used as template in a PCR amplification that used 5′-GGGATACATATGGAATATTCGG-3′ (upstream) and 5′-GCGAATTCTTAATAGAATCTTAG-3′ (downstream) oligonucleotides as primers. NdeI and EcoRI restriction sites were introduced in the primers to insert the amplified 1287-bp DNA fragment into a pET22b expression plasmid (Novagen), which was used to transform E. coli HMS174 (DE3) cells (Novagen). The nucleotide sequence of the insert was determined to confirm that no mismatching had occurred during the PCR amplification. Expression and Purification—A 100-ml overnight culture of E. coli HMS174 (DE3) cells, transformed with the mjSHMT overexpressing plasmid, was inoculated into 3 liters of Luria-Bertani broth containing ampicillin (100 mg/liter) and grown aerobically at 37 °C to exponential phase (A 600 = 0.3–0.4). Expression of mjSHMT was then induced with 0.2 mm isopropyl thio-β-d-galactopyranoside. After 20 h, cells were harvested and suspended in 100 ml of 50 mm Tris/HCl, 1 mm EDTA, pH 7.6. Cell lysis was obtained by the addition of 1 mg of lysozyme per g of packed bacterial cells. After incubation at 25 °C for 30 min, the cells were frozen overnight at –20 °C. After thawing, streptomycin sulfate (10 g/liter) was added to precipitate DNA. The was then at g for 30 min, and the was sulfate was added to the to The was at g for 20 min, and the was The was onto a phenyl-Sepharose with 50 mm pH containing sulfate to The was with 100 ml of the and the was a from the to 20 mm pH containing SHMT, and to a of 20 were for at °C to precipitate most of the After at g for 30 min, the containing mjSHMT and a few other was onto a DEAE-Sepharose that had been with 20 mm pH The was with 100 ml of the and the was then with a of in 20 mm pH were and as in the The of the mjSHMT was by and to be The was determined the at and a = to Anal. Biochem. PubMed Scopus Google by of the enzyme was by at 20 °C on a at a of with 20 mm pH containing by a was at and The was with = = aldolase = = = = and = of the purified SHMT was by were at 20 °C on a with The was in the of 0.2 to were at were at at a of with in a and were with the P. P. 2000; PubMed Scopus Google Scholar). were to were at and were at at a of with in a of was by to were with and with provided by E. J. PubMed Scopus Google Scholar). from were for to a a to a the was at the value determined from the The were in 20 mm pH and in 20 mm pH containing and 100 serine and H4MPT as the of was determined by this compound to and methylene-H4MPT dehydrogenase from M. extorquens The of the reaction, carried in mm pH at 37 was from the at of and a value of = L. Thauer J. Bacteriol. PubMed Google Scholar). The procedure was employed H4PteGlu was the pteridine that the enzyme employed in the assay was the bifunctional methylene-H4PteGlu dehydrogenase/methenyl-H4PteGlu cyclohydrolase from E. and the used in the was (8Schirch V. Hopkins S. Villar E. Angelaccio S. J. Bacteriol. 1985; 163: 1-7Crossref PubMed Google Scholar). The reaction mjSHMT at H4MPT was the pteridine or H4PteGlu was were determined to or in the of the of one substrate the was The of the of the reaction on H4MPT was determined at 30 mm and H4PteGlu between and was mm serine was between and 30 The was of the reaction at with of in 1 The was in 1 and used to the in at of the of a this a obtained previously a = were carried in 20 mm pH The of was by the reaction with of the by and thermostable alcohol dehydrogenase from brockii V. J. PubMed Google Scholar). The of the reaction was from the of in at a value of = from was at a value of = PubMed Scopus Google Scholar). The of with both was determined to Schirch V. PubMed Scopus Google and were the of were carried at 20 °C with a a were from with the at was between and 600 at and were at 20 °C in a were in a and and were in a The results are expressed as the a of per In all the and 100 were added were with a with a at 20 °C. at were at 20 °C with of in 20 mm pH or in 20 mm pH in the of and 100 After h, a that was to be to were at 20 °C. the of the the was at 20 °C with in 20 mm pH in the of mm and mm After h, the was by a with the used for the containing The was After h, a that was to be to were at 20 °C. from were by the singular value decomposition J. Scopus Google Scholar, J.C. 2000; PubMed Scopus Google Scholar, PubMed Scopus Google the is to the of in a of and to the and the in the were in a of one for in the The is by into the of = and are and is a The of the and those of the the of and are in of their of the of as by the of the singular in S. The the singular that the of in and V. important of is that the is very in the of and and the is in the and The induced by are by the of V. the of the of the information the was by and to a PubMed Scopus Google to is the of for a is the of in the absence of and is a that the in per of is the is the and is the for 1) The the as a function of in is the and are the and and are the and is the the is the of in the absence of is the of a is the and is the The is the at the of the to is as in of mjSHMT and was on of bacterial from liters of purification of the enzyme with a of mg per of culture was The purified enzyme a on with on as a and mjSHMT, on the sequence is in 20 mm pH from a with to a of suggesting that the enzyme is a at of SHMT of the enzyme was at 37 H4MPT or H4PteGlu as pteridine substrate. a was by the use of methylene-H4MPT dehydrogenase from M. extorquens AM1 as the enzyme in the which was enzyme with and 50 of pteridine could only be The of the of the reaction on to the with of as in I. The addition of 20 mm to the reaction not activity, from that reported for the enzyme from M. marburgensis (4Hoyt J.C. Oren A. Escalante-Semerena J.C. Wolfe R.S. Arch. Microbiol. 1986; 145: 153-158Crossref Scopus (22) Google Scholar). The was similar for the and that, the reaction is not by of the by from in which the of one substrate was whereas the substrate was at from in which the of one substrate was whereas the substrate was at not as reported in the were carried at from in which the of one substrate was whereas the substrate was at not as reported in the were carried at °C. in a could be also H4PteGlu, only if the enzyme was present in the assay at a much In this the of the on the pteridine substrate was on a from to H4PteGlu could not be employed with the not The with H4PteGlu could be the between substrate and is evidence that the of the assay and the be to = H4PteGlu is to with Schirch L. J. PubMed Google Scholar). was a possibility that the SHMT reaction with H4PteGlu was of the of by the between the and the folate to form 5,10-methylene-H4PteGlu. this the the in the absence of H4PteGlu. could be that the SHMT reaction H4PteGlu on the The of H4PteGlu to the enzyme was also by the of a to a J. PubMed Google Scholar), 0.2 mm H4PteGlu with enzyme and mm glycine at 37 °C in 20 mm pH a was mm H4MPT with enzyme and mm glycine not The enzyme was also of the of and to glycine and the of mm of or to the enzyme in a reaction, as by the of at and the of a with a at The to a which is with the E. coli enzyme Schirch V. PubMed Scopus Google Scholar), is in the reaction catalyzed by The of both which were carried at to the of two processes. the phase a = and was for of the whereas the phase had a = the phase to a = and of the the phase had a = Although both with first the reaction of is also with this enzyme is with and with of of the of of was or mjSHMT as the from 25 to °C (Fig. The of the reaction was both mm and at of mm The reaction catalyzed by mjSHMT was found to have °C that catalyzed by the E. coli with the substrate had a on the of both were used in a to the in which the to be to was a (Fig. of mjSHMT was by in the of at was present at 20 that the enzyme structure was not not were results to the of at with the aim of the for and reversible pH a is as by with a structure similar to that of the enzyme at pH to the of on mjSHMT at pH induced structural on the as by and the was with a at (Fig. The was as by the of the of the with and to and The at induced by of were of the and the by The in the from to were by SVD, which that only two to the The most significant singular are 0.2 and All the other singular are of the singular value and to of the first and the of the and as a function of not with those by the and The and of the enzyme at pH was and with that at pH with the aim of structural by the pH to the The at pH is of a (Fig. pH the of the is characterized by a similar to that at pH with the and and a of with that at pH (Fig. In the the of the enzyme at pH is in with the enzyme at pH and the is (Fig. pH the and present in the of the at pH and to the are (Fig. The at pH a of the at with that of the at pH (Fig. The of the at pH and at are similar and show the at (Fig. pH as with other enzymes in the M. Biochem. PubMed Scopus Google Scholar, M. B. PubMed Scopus Google Scholar, S. M. Biochem. PubMed Scopus Google Scholar), at or with at and (Fig. and transfer at is at (Fig. pH the of the (Fig. and at and are at the although a of the is at pH the transfer is (Fig. The of the structure in by G. Google the on the at pH and was at and to and and and and of other The of and was also with that determined by P. F. PubMed Scopus Google and 3 J. Google Scholar). results that at pH the SHMT structure is similar to that at pH structure to B. PubMed Google and The properties of the at pH that the of the into is by of the as by the and and suggest that the structure of the is similar to that of the value of for the enzyme at pH and at pH to of 100 and for with a J. J. Google Scholar). such of at pH and at pH were to a very was obtained for a at both pH the that the is at pH and into at pH SHMT has been isolated from several and from both the structural and functional of V. M. Scholar). of the enzyme from eubacterial and eukaryotic in the form or in with and H4PteGlu have been PubMed Scopus Google Scholar, G. S. Schirch V. PubMed Scopus Google Scholar, S. G. Schirch V. J. 2000; PubMed Scopus Google Scholar, L. P. 2000; PubMed Scopus Google Scholar, V. A. P. J. PubMed Scopus Google Scholar). a of the which form the active site and are involved in cofactor and substrate is on the structural features of archaeal SHMTs proposed that the of the cofactor in these enzymes no significant with respect to their bacterial and eukaryotic counterparts (3Paiardini A. Gianese G. Bossa F. Pascarella S. Proteins. 2003; 50: 122-134Crossref PubMed Scopus (30) Google Scholar). SHMT is characterized by very reaction and substrate In addition to its hydroxymethyltransferase activity, this enzyme is to in the absence of the pteridine a of such as the of and the of both V. M. Scholar). results show that the catalytic properties of mjSHMT those of the enzyme purified from other and confirm that the catalytic of SHMT is conserved the sequence of eubacterial and archaeal SHMTs is on the sequence of that all with are (Fig. and the that to the and of PLP, and which appear to the between and the of PLP, and and which with the of The involved in the of the are also and that the are from the other which appear to with the of the serine and and which bind to the of the On the other hand, the of mjSHMT toward H4MPT the of a site of the pteridine substrate and clearly represents a H4PteGlu also bind to mjSHMT and take part to the hydroxymethyltransferase reaction, although This that significant between and SHMTs be limited to only the pteridine have that the pteridine substrate to the enzyme from eubacterial and eukaryotic with similar although the and for the is for of the so far S. G. Schirch V. J. 2000; PubMed Scopus Google Scholar, L. P. 2000; PubMed Scopus Google Scholar, V. A. P. J. PubMed Scopus Google Scholar, T.F. G. Schirch V. J. 2003; PubMed Scopus Google Scholar). which to and of H4PteGlu, is the most important structural in the pteridine important is by which to the of H4PteGlu. are by the of and The portion of the active site that the pteridine the of the that is similar in all pteridine is to be structurally similar in all SHMTs. is (Fig. is present in all those to SHMT from which have a at this this does not to be related to the of the pteridine SHMTs use a modified folate pteridine is to that of H4MPT (2Maden B.E. Biochem. J. 2000; 350: 509-529Crossref Google Scholar). of the for the pteridine substrate is in the structure of SHMTs from methanogens that use H4MPT that SHMT is in is J. Microbiol. PubMed Scopus Google with respect to the eubacterial and eukaryotic enzymes (Fig. be related to the of these to a Moreover, the available of SHMT in with H4PteGlu do not any of the portion of the pteridine bind to the and not structural involved in this (3Paiardini A. Gianese G. Bossa F. Pascarella S. Proteins. 2003; 50: 122-134Crossref PubMed Scopus (30) Google Scholar). M. jannaschii is and is that the hydroxymethyltransferase reaction catalyzed by the of by both and and H4MPT or H4PteGlu as pteridine substrate. the of does not to the of the The of the enzyme in the aldolase reaction that with mjSHMT or the with substrate has a (Fig. This is in with previously reported on the of and substrate analogues with SHMT, which that the of results in a of the enzyme V. M. M. J. 1991; PubMed Google Scholar). The of the aldolase reaction from the to be the for both these results confirm that mjSHMT is similar in several to the enzyme from mesophilic sources. mjSHMT is to pH the a structure similar to the form with a significant of the as by the and and the by of the This that play a significant role in the of the The of the structure is the reported for many from whereas the is at a value similar to that reported for the E. coli enzyme at pH Schirch Schirch V. J. PubMed Scopus Google Scholar). This that the of a at 20 °C. the resistance of the enzyme is by the of a significant of structure in the of at pH the of mjSHMT at pH be a a PubMed Scopus Google Scholar). structural between SHMT from mesophilic and carried the of homology that the thermal in SHMT be the of the of increased of at the and increased at the (3Paiardini A. Gianese G. Bossa F. Pascarella S. Proteins. 2003; 50: 122-134Crossref PubMed Scopus (30) Google Scholar). The results obtained with mjSHMT with these Schirch and Rolf Thauer for during the of the and for are to Schaffhausen, for kindly H4PteGlu.