-methylglucosyl-containing lipopolysaccharides (MGLPs; Lee & Ballou, 1964
; Lee, 1966
) and the 3-O
-methylmannosyl-containing polysaccharides (MMPs; Gray & Ballou, 1971
; Maitra & Ballou, 1977
) are two unusual polymethylated polysaccharides (PMPS) produced by mycobacteria. Both PMPS localize to the cytoplasm, where they have been proposed to regulate fatty-acid biosynthesis owing to their ability to form stable 1:1 complexes with long-chain fatty acids and acyl-coenzyme A derivatives. In sequestering the products of fatty-acyl synthase I (FAS I), PMPS are thought to facilitate the release of the neo-synthesized chains from the enzyme, thereby not only reopening active sites essential for enzyme turnover but also terminating their elongation (for a review, see Bloch, 1977
). In addition, PMPS have been proposed to serve as general fatty-acyl carriers, the role of which would be to facilitate the further processing of very long and insoluble fatty-acyl CoAs, including mycolic acids, by increasing the tolerance of mycobacteria to high cytoplasmic concentrations of these products while protecting them from degradation (Yabusaki & Ballou, 1979
). The MGLPs of Mycobacterium bovis
BCG are composed of ten α-(1→4)-linked 6-O
-methylglucosyl residues with a nonreducing end made of the tetrasaccharide 3-O
-α-(1→, whereas the tetrasaccharide →4)-[α-(1→4)-d
-α(1→ linked to position 2 of d
-glyceric acid constitutes the reducing end of the molecule. Positions 3 of the second and fourth α-d
residues (closest to the reducing end) are substituted by single β-d
residues. The nonreducing end of the polymer can be acylated by a combination of acetate, propionate and isobutyrate. The Glcp
residues of the reducing end can be esterified with up to three succinate groups and position 1 of glyceric acid can also be esterified by octanoate. MGLPs occur in the cell as a mixture of four main components that differ in their content of esterified succinic acid (Tuffal et al.
). Although the structures of MGLPs and their fatty-acyl-binding properties have been well established, little is known about the biosynthesis of these important molecules.
Empadinhas and coworkers recently showed that recombinant forms of the GpgS enzymes of M. bovis
BCG and M. smegmatis
display glucosyl-3-phosphoglycerate activity in vitro
, transferring a Glcp
residue from UDP-Glc to the 2 position of d
-3-phosphoglycerate (PG) to form α(1→2)-d
-3-phosphoglycerate (GPG; Empadinhas et al.
). Furthermore, recent evidence based on the analysis of an M. smegmatis
knockout mutant deficient in the expression of gpgS
clearly implicated this enzyme in the transfer of the first Glcp
residue of MGLP, generating GPG. This product is subsequently dephosphorylated by an unknown phosphatase to yield the glucosyl-glycerate (GG) subunit found at the reducing end of MGLP (Kaur et al.
, manuscript in preparation). A second and third Glcp
residue are subsequently added by unidentified glucosyltransferase/branching enzymes to form [α-(1→4)-d
-glyceric acid. Interestingly, a cluster of genes dedicated to the biosynthesis of MGLP has recently been identified in M. smegmatis
and M. tuberculosis
(Stadthagen et al.
). It was demonstrated that Rv3032
encodes the main α-(1→4)-glucosyltransferase responsible for the elongation of MGLP, whereas Rv3030
encodes the O-methyltransferase likely to be required for the 6-O-methylation of these lipopolysaccharides.
Glycosyltransferases (GTs) can be classified into either ‘inverting’ or ‘retaining’ enzymes according to the anomeric configuration of the reaction substrates and products. A single-displacement mechanism is well established for ‘inverting’ enzymes, whereas the catalytic mechanism for ‘retaining’ enzymes, including GpgS, remains unclear (Empadinhas et al.
; Lairson et al.
). Interestingly, only two protein topologies have been found for the nucleotide sugar-dependent enzymes from the first 29 GT sequence-based families (CAZy, Carbohydrate-Active Enzymes Database; see http://www.cazy.org
) for which three-dimensional structures have been reported. These topologies are variations of ‘Rossmann-like’ domains and have been defined as GT-A (Charnock & Davies, 1999
) and GT-B (Vrielink et al.
). Both inverting and retaining enzymes have been found within the GT-A and GT-B fold GTs, indicating that there is no correlation between the overall fold of GTs and their catalytic mechanism. GpgS belongs to the recently defined GT81 family of GTs. The crystallographic characterization of GpgS will shed light on the catalytic mechanism of the GT81 family of GTs and the biosynthesis of MGLP in mycobacteria.