Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus

Fratte, Sonia Delle; White, Robert H.; Maras, Bruno; Bossa, Francesco; Schirch, Verne

doi:10.1128/jb.179.23.7456-7461.1997

Cited by 17 publications

(11 citation statements)

References 15 publications

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“…Besides catalyzing its main physiological reaction of SHMT activity, the enzyme is known to catalyze a broad range of reactions, including a retro-aldol cleavage of 3-hydroxy-amino acids, transamination and decarboxylation [7,8]. Although SHMTs from many prokaryotes and eukaryotes have been studied [11][12][13][14][15][16][17], in-depth investigation has focused only on SHMTs from rabbit and sheep liver, as well as E. coli and Bacillus stearothermophilus [6,7,11,18,19].…”

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confidence: 99%

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

et al. 2009

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The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 m−1·cm−1. The Kd for binding of the enzyme and pyridoxal‐5‐phosphate was 0.14 ± 0.01 μm. An alternative assay for measuring the tetrahydrofolate‐dependent SHMT activity based on the coupled reaction with 5,10‐methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary‐complex mechanism with a kcat value of 0.98 ± 0.06 s−1 and Km values of 0.18 ± 0.03 and 0.14 ± 0.02 mm for l‐serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius’s plot showed a transition temperature of 19 °C. Besides l‐serine, PvSHMT forms an external aldimine complex with d‐serine, l‐alanine, l‐threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate‐independent retro‐aldol cleavage of 3‐hydroxy amino acids. Although l‐serine is a physiological substrate for SHMT in the tetrahydrofolate‐dependent reaction, PvSHMT can also use d‐serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme–quinonoid complex with d‐serine, but not with l‐serine, whereas SHMT from rabbit liver was reported to form an enzyme–quinonoid complex with l‐serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.

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confidence: 99%

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

et al. 2009

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“…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 (4), recently renamed Methanothermobacter marburgensis (5), and from Sulfolobus solfataricus (6), 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.…”

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confidence: 99%

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

Angelaccio¹,

Chiaraluce²,

Consalvi³

et al. 2003

Journal of Biological Chemistry

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The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of C␤ of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C 1 ) units for several essential biosynthetic processes. In many Archaea, C 1 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, 1 EC 2.1.2.1) catalyzes the reversible transfer of C␤ of serine to tetrahydropteroylglutamate (H 4 PteGlu) to form glycine and 5,10-methylene-H 4 PteGlu. In Eukarya and Eubacteria, H 4 PteGlu functions as a carrier of C 1 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 C 1 units onto the folate carrier (1). In methanogens and several other Archaea, C 1 fragments from formyl to methyl oxidation levels are carried by tetrahydromethanopterin (H 4 MPT), a pterin-containing compound involved in methanogenesis. Although H 4 PteGlu and H 4 MPT are structurally similar in their pterin-like portion (Fig. 1) and in the role as C 1 units carriers, they are functionally distinct. H 4 MPT does not appear to be suited to most of the biosynthetic functions of H 4 PteGlu. Moreover, the biosynthetic pathways of the two carriers have few, if any, homologies, suggesting the possibility of separate evolutionary origins (2). In the metabolism of folates, SHMT represents a...

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“…volcanii seems to be the exception among the archaea. In the methanogenic and thermoacidophilic archaea, C 1 transfer reactions have been shown to be carried out either with modified folates or in the absence of folate derivatives (Nyce and White, 1996; Delle Fratte et al ., 1997; White, 1997b). Moreover, inspection of the five complete archaeal genome sequences published to date did not reveal sequence homologies to DHFR or to bacterial and eukaryotic thymidylate synthases.…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have shown that considerable amounts of folates exist in the extremely halophilic archaea, whereas the methanogenic and thermoacidophilic archaea contain, in their place, a variety of modified folates (White, 1988; 1991; 1997a; Worrell and Nagle, 1988). In some archaea, purine biosynthesis requires neither folates nor modified folates (White, 1997b), whereas the biosyntheses of thymidine (Nyce and White, 1996) and glycine (Delle Fratte et al ., 1997) involve only modified folates.…”

Section: Introductionmentioning

confidence: 99%

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

Ortenberg

Rozenblatt-Rosen²,

Mevarech³

2000

Molecular Microbiology

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SummaryThe gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DhdrA, DhdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.

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Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus

Cited by 17 publications

References 15 publications

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

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

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

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