Identification of the mycobacterial glucosyl-3-phosphoglycerate synthase

Abstract

Mycobacteria synthesize unique polysaccharides that regulate fatty acid synthesis, namely the methylglucose lipopolysaccharide (MGLP) and the methylmannose polysaccharide. Glucosyl-(1-->2)-glycerate is found at the reducing end of MGLP. The mycobacterial gene encoding a glucosyl-3-phosphoglycerate synthase (GpgS), primarily found in actinobacteria and sharing very low amino acid identity with known homo-functional GpgSs, has been identified. This gene has been annotated as an inverting family 2 glycosyltransferase of unknown function. The gpgS genes from the fast-growing Mycobacterium smegmatis strain 1102 and from the slow-growing Mycobacterium bovis BCG in Escherichia coli were expressed, and the recombinant enzymes were purified and characterized. The substrates for optimal activity were UDP-glucose and d-3-phosphoglycerate but ADP-glucose was also an efficient donor. The enzymes had maximal activity around 45 degrees C, pH 8.0, and were strictly dependent on Mg(2+). In Mycobacterium tuberculosis H37Rv, the gene encoding GpgS (Rv1208) is identical to the homologue in Mycobacterium bovis BCG and was considered to be essential for growth. It is shown that these genes encode retaining family 81 glycosyltransferases regardless of the low amino acid identity with other known enzymes of this family.

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

“…The enzyme from M. burtonii was specific for GDP-glucose and despite the similarity of this substrate with GDP-mannose and of the reaction mechanism, GpgSs and MpgSs shared no sequence identity, save for short motifs. Remarkably, this GpgS also had very low amino acid sequence identity with the homo-functional GpgSs from mycobacteria [18]. On the other hand, the recombinant MpgP from M. burtonii dephosphorylated GPG to GG and MPG to MG. Due to the vicinity with the gpgS gene, it was designated gpgP.…”

“…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a highly divergent MpgS from which sequence homology to the known MpgSs had been extensively erased (Empadinhas et al, unpublished results).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.…”

“…The biosynthesis of GG in mycobacteria as precursor for the MGLP The identification of GG in the mycobacterial MGLP, as well as detection of homologues (40% amino acid identity) to the mpgS from R. xylanophilus in mycobacterial genomes, led to the study of the genes from two species from which the MGLPs have been studied, the fast-growing M. smegmatis and the slow-growing M. bovis BCG [18,28,50]. The corresponding recombinant Fig.…”

“…The gpgS gene was present in all the mycobacterial genomes available and located in a conserved operon-like structure containing genes folP and tagA, encoding a putative dihydropteroate synthase (DHPS) and a DNA glycosylase, respectively. Despite the fact that GG has been detected in M. smegmatis extracts no typical phosphatase gene (gpgP) has been detected in mycobacterial genomes suggesting that the dephosphorylation of GPG may reflect excess GPG that is not used for MGLP synthesis or is converted into GG by unspecific phosphatases/hydrolases [18].…”

Genome-Wide Relative Analysis of Codon Usage Bias and Codon Context Pattern in the Bacteria Salinibacter Ruber, Chromohalobacter Salexigens and Rhizobium Etli

“…There is a wide range of halophilic microorganisms which comprise domains of Archaea and Bacteria. The saline cytoplasm of these bacteria requires enzymes which are rich in acidic amino acids and dependent on K+ or Na+ for their biological activity [3]. Oren and Mana 2002 [4] have reported that these organisms include: (i) the extremely halophilic Archaea of the family Halobacteriaceae, which comprises Halobacterium, Haloarcula, Haloquadratum, Halorhabdus, Natronobacterium and Natronococcus (ii) the halophilic Bacteria of the order Haloanaerobiales and (iii) the bacterium S. ruber.…”