Abstract
The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of Cbeta 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.
Highlights
Teroylglutamate (H4PteGlu) to form glycine and 5,10-methylene-H4PteGlu
The purified enzyme exhibited a single band on SDSPAGE when stained with Coomassie blue and, on analytical ultracentrifugation, sedimented as a single, sharp, and symmetrical peak. mjSHMT, whose predicted subunit size based on the amino acid sequence is 48.2 kDa, eluted in 20 mM sodium phosphate, pH 7.2, from a calibrated size-exclusion chromatography column (Superose 12) with an elution volume corresponding to a protein of 98 kDa, suggesting that the enzyme is a dimer at neutral pH
A multiple sequence alignment of eubacterial and archaeal serine hydroxymethyltransferase (SHMT) shows that all residues interacting with PLP are invariant (Fig. 6): His126 and Ala202, the residues that stack, respectively, to the re face and si face of PLP, His129 and Asn102, which appear to stabilize the crucial interaction between Asp200 and the pyridinium nitrogen of PLP, and His203 and Ser 175, which interact with the phenol oxygen of PLP pyridine ring
Summary
Teroylglutamate (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. 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 [3], other enzymes that use H4MPT as cofactor do not show any significant homology to their eukaryotic and eubacterial counterparts [2]. Modified folates are not commercially available, and this has clearly hindered a satisfactory characterization of archaeal SHMTs. the purification of the enzyme from Archaea that thrive in extreme environments is complicated by the difficulty of growing these organisms in a laboratory. The enzyme was proposed to function in vivo in the direction of serine biosynthesis 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 [2, 7]. The aim of the research was to assess the extent to which mjSHMT is structurally and mechanistically similar to its prokaryotic and eukaryotic counterparts
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