Characterization and Regulation of the Osmolyte Betaine Synthesizing Enzymes GSMT and SDMT from Halophilic Methanogen Methanohalophilus portucalensis

Abstract

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize the osmolyte betaine de novo in response to extracellular salt stress. Betaine is generated by the stepwise methylation of glycine to form sarcosine, N, N-dimethylglycine and betaine by using S-adenosyl-L-methionine (AdoMet) as the methyl donor. The complete gene cluster of Mpgsmt-sdmt was cloned from Southern hybridization and heterologous expressed in E. coli respectively. The recombinant MpGSMT and MpSDMT both retained their in vivo functional activities in E. coli BL21(DE3)RIL to synthesize and accumulate betaine and conferred elevated survival ability in betaine transport deficient mutant E. coli MKH13 under high salt stress. The dramatic activating effects of sodium and potassium ions on the in vitro methyltransferase activities of MpGSMT, but not MpSDMT or bacterial GSMT and SDMT, revealed that GSMT from halophilic methanoarchaeon possesses novel regulate mechanism in betaine biosynthesis pathway. The circular dichroism spectra showed the fluctuated peaks at 206 nm were detected in the MpGSMT under various concentrations of potassium or sodium ions. This fluctuated difference may cause by a change in the β-turn structure located at the conserved glycine- and sarcosine-binding residue Arg167 of MpGSMT. The analytical ultracentrifugation analysis indicated that the monomer MpGSMT switched to dimeric form increased from 7.6% to 70% with KCl concentration increased from 0 to 2.0 M. The level of potassium and sodium ions may modulate the substrate binding activity of MpGSMT through the conformational change. Additionally, MpGSMT showed a strong end product, betaine, inhibitory effect and was more sensitive to the inhibitor AdoHcy. The above results indicated that the first enzymatic step involved in synthesizing the osmolyte betaine in halophilic archaea, namely, GSMT, may also play a major role in coupling the salt-in and compatible solute (osmolyte) osmoadaptative strategies in halophilic methanogens for adapting to high salt environments.

Structural Analysis of Glycine Sarcosine N-methyltransferase from Methanohalophilus portucalensis Reveals Mechanistic Insights into the Regulation of Methyltransferase Activity

“…The pronounced sequence similarity between Mp GSMT and GNMT agrees with their overlapping activity as follows: both enzymes catalyze the SAM-dependent methylation of glycine to produce sarcosine2425. Structural analysis revealed that, similarly to all Class I methyltransferases, the catalytic core of GNMT adopts the Rossmann fold, which bears the SAM and substrate binding pockets262728.…”

“…However, there are outstanding distinctions between the two enzymes. First, while Mp GSMT is negatively regulated by the end product betaine25, the activity of GNMT is regulated by folate1430. Second, both glycine and sarcosine can be methylated by Mp GSMT25, whereas GNMT only acts on glycine3132.…”

Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

“…The synthesis pathway that rests on the methylation of glycine does not require the uptake of a precursor molecule and yields the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG) ( Fig. 1) (27,(30)(31)(32)(33).…”

Linking microbial metabolism and organic matter cycling through metabolite distributions in the ocean

“…2). Betaine and DMSP are both osmolytes, which are molecules present in the cytosol of cells that regulate osmotic pressure as well as assist in maintaining protein structure (Yancey et al 1982).. Betaine, by far the largest contributor to the metabolite composition of the sinking particles, is an osmolyte utilized by organisms across the domains of life, although it is less commonly used in phytoplankton than DMSP (Yancey et al 1982;Boch et al 1997;Keller et al 1999;Lai and Lai 2011). Betaine concentrations can reach 170 mM inside a phytoplankton cell (Keller et al 1999), suggesting that this metabolite is likely to be measured at relatively high concentrations in particulate samples.…”