Consequently , it is of interest to purify native GSMT fromMethanohalophilus portucalensisto further compare its enzymatic characteristics and kinetics with rGSMT

Consequently , it is of interest to purify native GSMT fromMethanohalophilus portucalensisto further compare its enzymatic characteristics and kinetics with rGSMT. monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited byS-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 Xphos 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 Xphos 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. == Introduction == The halophilic methanoarchaeonMethanohalophilus portucalensisFDF1Tis F2rl3 a model strain for investigating the strategy of acclimation under salt and temperature stresses because it was isolated from a solar saltern, where it has a wide range of temperature gradients and salt concentrations [1]. One strategy used to adapt to salt and osmotic stresses is the accumulation of potassium ions and small molecular osmolytes (compatible solutes), including -amino acid derivatives, -glutamate, -glutamine, and N-acetyl–lysine, which are observed ubiquitously in methanoarchaea [24]. The osmolyte glycine betaine (betaine) possesses the highest osmoprotective efficiency and can be taken up as a compatible solute to encounter salt, osmotic and cold stresses among three organismal domains [3, 57]. Betaine is actively transported via BtaABC (betainetransporter inarchaea) [8], and two open reading frames of a sodium/proton forced choline/carnitine/betaine transporter were identified in theM. portucalensisFDF1Tgenome (Lai et al., unpublished data). Additionally , betaine can be synthesized from its precursor, glycine, via a three-step methylation process with S-adenosylmethionine (SAM), a methyl donor, by glycine sarcosineN-methyltransferase (GSMT, AEG64703) and sarcosine dimethylglycineN-methyltransferase (SDMT, AEG64704) to produce the intermediates sarcosine and dimethylglycine; the end product, betaine; and a competitive inhibitor, S-adenosylhomocysteine (SAH) [813]. Although many halophilic or halotolerant bacteria can synthesize betaine by a two-step oxidation pathway, only extreme halophilic bacteria, includingActinopolyspora halophila, Halorhodospira halochloris, Aphanothece halophytica, non-halophilicSynechococcussp. WH8102, andMyxococcus xanthus, possess GSMT and SDMT/DMT, which are required for betaine synthesis [1418]. Our previous studies showed thatM. portucalensisFDF1Tpossesses two betaine synthesis enzyme systems, GSDMT [10] and GSMT/SDMT [9, 11], which are unique among all known isozymes from halophilic/halotolerant bacteria. It is noteworthy that GSDMT fromM. portucalensisis the only amineN-methyltransferase with broad specificity for the substrates glycine, sarcosine, and dimethylglycine [10], whereas the other system exhibits partially overlapping Xphos substrate specificity between GSMT and SDMT/DMT [1418]. The activity of recombinant MpGSMT (rGSMT) is activated by increasing monovalent ions to adapt to osmotic stress and is feedback-inhibited by the end product betaine to conserve energy [11]. Recombinant MpSDMT (rSDMT) is a constitutive enzyme used to produce the osmolyte betaine, which is limited by the amount of the substrate, sarcosine, and the inhibitor, SAH [11]. Recently, phosphoproteomic analysis ofM. portucalensisFDF1Tshowed that both GSMT and SDMT are post-translationally modified by phosphorylation [19]. According to our previous data, native SDMT showed lower substrate binding affinity than rSDMT, whereas the reaction velocity of SDMT was higher than that of rSDMT [9, 11]. We hypothesize that this phenomenon may be due to the substrate specificity of protein kinases fromM. portucalensisFDF1Tand the recombinant hostEscherichia coli, which causes different levels of phosphorylation between SDMT and rSDMT. In this study, we purified native GSMT fromM. portucalensisFDF1Tfor enzymatic comparison with rGSMT. The phosphorylation sites of GSMT and purified rGSMT were identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the different modifications between native GSMT and rGSMT. == Materials and Methods == == Organism and growth conditions == The halophilic methanoarchaeonM. portucalensisFDF1T(DSM 7471) was routinely cultured in H-P-defined medium containing 2 . 1 M NaCl and 20 mM trimethylamine as the carbon and energy source, respectively [1, 3]. The medium was prepared under a N2: CO2atmosphere (4: 1) to eliminate oxygen. Methanohalophilus portucalensisFDF1Tcultures were inoculated at a.