title: Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T.
abstract: The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea.
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Institute of Biological Chemistry, Academia Sinica. Taipei, Taiwan.
authors.forename: Shu-Jung
authors.initials: SJ
authors.lastname: Lai
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Deng
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Mei-Chin
authors.initials: MC
authors.lastname: Lai
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D050938:Glycine N-Methyltransferase / Q000378:metabolism*; D007700:Kinetics; D017019:Methanosarcinaceae / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001622:Betaine; C025138:dimethylglycine; D012435:S-Adenosylhomocysteine; D050938:Glycine N-Methyltransferase; D005998:Glycine; D012521:Sarcosine