title: 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.
authors.affiliation: Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università di Napoli, Via Costantinopoli 16, 80138, Naples, Italy, marina.porcelli@unina2.it.
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
authors.forename: Concetta Paola
authors.initials: CP
authors.lastname: Ilisso
authors.forename: Ester
authors.initials: E
authors.lastname: De Leo
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D008715:Methionine; D008025:Ligases