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

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

The Phylum dictyoglomi

The phylum Dictyoglomi consists a single genus, Dictyoglomus, with two type strains and several related pure culture isolates. All isolates are thermophilic anaerobic Gram-type negative rods. A major distinguishing phenotypic feature is the formation of spherical bodies in late stationary phase of growth, the function of which is not understood.Most isolates are fermentative using a range of simple carbohydrates, but some isolates are able to grow on crystalline cellulose and chemolithotrophy using carbon monoxide as energy source has been reported for one pure culture. There have been relatively few applications for Dictyoglomus enzymes as a result of a number of factors. Although many of their kinetic properties are exceptional, they have had to be cloned and expressed in standard fermentation strains as hosts, and their low G:C content has required significant genetic manipulation to provide expression. Some of the main applications have required inexpensive enzymes in bulk (e.g., pulp bleaching in paper manufacture), and they have had to be regarded as a replacement for well-established enzymes currently used in the industry. The major applications have involved glycosyl hydrolases, but new uses in value-added products involving drug precursor transformations have been reported recently.10 page(s