Biochemical Characterization of a Thermostable Adenosylmethionine Synthetase from the Archaeon Pyrococcus Furiosus with High Catalytic Power

Abstract

Adenosylmethionine synthetase plays a key role in the biogenesis of the sulfonium compound S-adenosylmethionine, the principal widely used methyl donor in the biological methylations. We report here, for the first time, the characterization of adenosylmethionine synthetase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMAT). The gene PF1866 encoding PfMAT was cloned and expressed, and the recombinant protein was purified to homogeneity. PfMAT shares 51, 63, and 82% sequence identity with the homologous enzymes from Sulfolobus solfataricus, Methanococcus jannaschii, and Thermococcus kodakarensis, respectively. PfMAT is a homodimer of 90 kDa highly thermophilic with an optimum temperature of 90 °C and is characterized by remarkable thermodynamic stability (Tm, 99 °C), kinetic stability, and resistance to guanidine hydrochloride-induced unfolding. The latter process is reversible as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. Limited proteolysis experiments indicated that the proteolytic cleavage site is localized at Lys148 and that the C-terminal peptide is necessary for the integrity of the active site. PfMAT shows kinetic features that make it the most efficient catalyst for S-adenosylmethionine synthesis among the characterized MAT from Bacteria and Archaea. Molecular and structural characterization of PfMAT could be useful to improve MAT enzyme engineering for biotechnological applications.

Regiocomplementary O‐Methylation of Catechols by Using Three‐Enzyme Cascades

“…Recently, the structural and functional characterisation of a variety of MATs from thermophilic Archaea has been achieved. These enzymes are promising candidates for biotechnological use, due to their enhanced stability . We chose the MAT from Thermococcus kodakarensis ( Tk MAT) that was characterised in earlier studies by our working groups to evaluate its suitability for application in methylation cascades.…”

“…These enzymes are promising candidates for biotechnological use, due to their enhanced stability. [26][27][28][29] We chose the MAT from Thermococcus kodakarensis (TkMAT) that was characterised in earliers tudies by our working groups [27] to evaluate its suitability for applicationinm ethylation cascades.…”

Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum

“…Interestingly, SAM synthetase is one of the most abundant proteins in the cold proteome (5th ranked, 0.97%), which provides the substrate for SAM-dependent methyltransferases. SAM synthetase generates the methyl group donor involved into polyamine synthesis, methylation of DNA and other organic molecules [48] that may contribute to the cold stress response in C. divulgatum. While the Cuniculiplasma genome encodes 12 SAM-dependent methyltransferase homologues (including one pseudogene), only three of them were detected in this proteome dataset: (i) CSP5_0484 (low-identity to isoaspartyl methylransferase from Archaeoglobus and 6% downregulated in the cold), (ii) CSP5_0705 (50% AA similarity with bacterial ubiquinone/menaquinone biosynthesis C-methyltransferases and arsenite methyltransferase in Methanosarcina, with exactly the same expression levels under both conditions) and (iii) CSP5_1828 (48% AA similarity with bacterial rRNA guanine or uracil methyltransferases, 4% up in the cold).…”

S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs

“…AdoMet is biosynthesized from l -methionine and ATP by methionine adenosyltransferase (MAT, EC 2.5.1.6.) in a two-step reaction in which the energy-rich sulfonium compound is formed by the dephosphorylation of ATP [ 4 – 6 ]. All living cells express MAT highlighting its essential role in regulating appropriate levels of AdoMet [ 7 ].…”