Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine <i>N</i>-Methyltransferase from <i>Methanohalophilus portucalensis</i> FDF1^T

<div><p>The halophilic methanoarchaeon <i>Methanohalophilus portucalensis</i> FDF1^T 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 <i>N</i>-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine <i>N</i>-methyltransferase (rSDMT) from <i>Escherichia coli</i> 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 <i>Methanohalophilus portucalensis</i> 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 <i>S</i>-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.</p></div

The yields and activities of GSMT purified from <i>Methanohalophilus portucalensis</i> FDF1^T.

<p>The yields and activities of GSMT purified from <i>Methanohalophilus portucalensis</i> FDF1^T.</p

Native GSMT was purified by employing ion exchange and size exclusion chromatography.

<p>(A) FPLC profiles of Hiprep DEAE FF 16/10 Sepharose chromatography with step and linear gradients of KCl concentrations. The solid line indicates the absorbance at 280 nm, whereas the dashed line displays the KCl concentration. D1 to D8 indicate the peak numbers. (B) FPLC profiles of Hiprep 16/10 Sephacryl S-200 HR gel filtration chromatography. G1 to G5 indicate the peak numbers. (C) GSMT-containing protein fractions were separated by 12.5% SDS-PAGE, followed by silver staining. (D) Estimation of GSMT molecular weight via gel filtration with low-molecular-weight standard proteins, including IgG (160 KDa), BSA (67 KDa), β-lactoglobulin (35 KDa), cytochrome C (12.4 KDa), and cytidine (0.24 KDa).</p

The reaction velocity of GSMT was significantly promoted under higher concentrations of KCl.

<p>Lineweaver-Burk plots of GSMT under various concentrations of KCl with glycine (A) or sarcosine (B) as the substrate. (C) Kinetic parameters of GSMT and rGSMT under 0.5 to 1.5 M KCl conditions. All data points were averaged from three independent experiments.</p

Kinetic parameters of betaine synthesizing enzymes from <i>M</i>. <i>portucalensis</i> FDF1^T.

<p>Kinetic parameters of betaine synthesizing enzymes from <i>M</i>. <i>portucalensis</i> FDF1^T.</p

Additional file 3: Figure S1. of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

The relative abundances of amino acids between −10 and +10 positions of the phosphorylated or non-phosphorylated His/Asp (PDF 377 kb)

Additional file 2: Table S1. of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

Phospho probabilities, process method, leading protein identifier, UniProt No. (if available), protein description, and protein functional class of the identified phosphopeptides (PDF 539 kb)

Additional file 1: of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

MS/MS Spectra. Spectra of the identified phosphopeptides (PDF 4714 kb)