Characterization of the Biosynthetic Pathway of Glucosylglycerate in the Archaeon Methanococcoides burtonii

Abstract

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and D-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50°C. The maximal activity of GpgP was at 50°C with GPG as the substrate and at 60°C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.Most microorganisms capable of osmotic adjustment accumulate low-molecular-weight organic compounds, commonly designated compatible solutes, which can be taken up from the environment or synthesized de novo (6). Although the uptake of organic solutes such as glycine betaine and trehalose, among others, by microorganisms under salt stress is preferred because it is energetically favorable (10), many organisms synthesize specific compatible solutes because these are not present in the environment or those that are present do not fulfill the specific requirements of the organisms.Ectoine, glycine betaine, trehalose, and glutamate are among the most common compatible solutes of bacteria and archaea, where they normally accumulate in response to stress imposed by salt (10). Mannosylglycerate (MG), di-myo-inositol-phosphate, and diglycerol-phosphate have a more restricted distribution, being found primarily in (hyper)thermophilic prokaryotes. Mannosylglycerate is widespread in (hyper)thermophilic bacteria and archaea and is also found in some red algae (21, 35). Cyclic 2,3-bisphosphoglycerate has only been encountered in methanogens (20,25,36).The rare solute glucosylglycerate (GG) was originally identified in the cyanobacterium Agmenellum quadruplicatum strain PCC7002 grown under nitrogen-limiting conditions (22). This solute was also detected in the archaeon Methanohalophilus portucalensis strain FDF...

To be or not to be a compatible solute: Bioversatility of mannosylglycerate and glucosylglycerate

“…Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from T. thermophilus, P. horikoshii, and ''D. ethenogenes'' [8].…”

“…Recent research on the biosynthesis of GG in the psychrotolerant archaeon Methanococcoides burtonii (M. burtonii) led to the hypothesis that GG could have a protective role when the organism grows under low nitrogen conditions. However, it was not possible to demonstrate this assumption since the organism did not grow in the absence of trimethylamine [8]. In spite of the occasional detection of free GG in a few organisms, the levels were too low to have any significant osmotic effect.…”

“…The purification of the native enzyme allowed the identification of a highly divergent MpgS from which sequence homology to the known MpgSs had been extensively erased (Empadinhas et al, unpublished results). Adjacent to this mpgS gene we detected genes encoding two unspecific phosphatases, leading to the hypothesis that one or both could complete the two-step synthesis of MG. On the other hand, this atypical MpgS had high homology with one enzyme found in mycobacteria and related Actinobacteria, later found to be a glucosyl-3-phosphoglycerate synthase (GpgS) with low homology to the homo-functional GpgSs from M. burtonii and P. marina [8,9,18]. In fact, the R. xylanophilus MpgS could synthesize GPG, the phosphorylated precursor of GG, with higher efficiency than that for MPG, although free GG has never been detected in R. xylanophilus.…”

“…In fact, GG has not been detected in P. miotherma in any of the conditions tested and the precursor for MGG biosynthesis has recently been identified to be glucosyl-3-phosphoglycerate (GPG) (Fernandes, unpublished results). This compound is the phosphorylated precursor for the GG found in several organisms [8,9].…”

“…A glucosyltransferase gene immediately upstream of the phosphatase was functionally characterized as a glucosyl-3-phosphoglycerate synthase (GpgS) [8]. The new family GT81 of glycosyltransferases was created to accommodate this enzyme and homologues (www.…”