Crystal Structure of a UDP-glucose-specific Glycosyltransferase from a Mycobacterium Species

Abstract

Glycosyltransferases (GTs) are a large and ubiquitous family of enzymes that specifically transfer sugar moieties to a range of substrates. Mycobacterium tuberculosis contains a large number of GTs, many of which are implicated in cell wall synthesis, yet the majority of these GTs remain poorly characterized. Here, we report the high resolution crystal structures of an essential GT (MAP2569c) from Mycobacterium avium subsp. paratuberculosis (a close homologue of Rv1208 from M. tuberculosis) in its apo-and ligand-bound forms. The structure adopted the GT-A fold and possessed the characteristic DXD motif that coordinated an Mn 2؉ ion. Atypical of most GTs characterized to date, MAP2569c exhibited specificity toward the donor substrate, UDP-glucose. The structure of this ligated complex revealed an induced fit binding mechanism and provided a basis for this unique specificity. Collectively, the structural features suggested that MAP2569c may adopt a "retaining" enzymatic mechanism, which has implications for the classification of other GTs in this large superfamily.

Structural Analysis of Thermus thermophilus HB27 Mannosyl-3-phosphoglycerate Synthase Provides Evidence for a Second Catalytic Metal Ion and New Insight into the Retaining Mechanism of Glycosyltransferases

“…Comparing the structures of MpgS, GpgS, and MgS, tunnel II (Fig. 4B) emerges as the most likely common candidate for acceptor entrance into the active site of MpgS as well as of MgS and GpgS, in agreement with previous proposals (9,42,43).…”

“…Secondary structure matching against all of the PDB using the DALI (41) search engine, revealed a general structural preservation with many other nucleoside-diphospho-sugar (NDP-sugar) transferases, including those involved in the synthesis of lipopolysaccharides, as well as some pyrophosphorylases present at the early stages of peptidoglycan biosynthesis. However, as expected, the highest Z-score (52.9) was observed for its orthologous family member P. horikoshii MpgS, followed by members of the GT81 family: Mycobacterium tuberculosis glucosyl-3-phosphoglycerate synthase (GpgS) (42) (PDB 3E26; Z-score ϭ 21.9), the putative GpgS from Mycobacterium avium paratuberculosis encoded by MAP2569c (PDB 3CKN; Z-score ϭ 21.8) (43), and the novel MpgS/GpgS from Rubrobacter xylanophilus that is able to use GDP-Man as well as GDP-glucose as substrates (PDB 3F1Y; Z-score ϭ 21.2) (44). This last enzyme is suggested to be at the evolutionary split from which all MpgSs and GpgSs have evolved (38).…”

“…-Gly 316 ), which are involved in the substrate binding-mediated response (9,42,43). In addition to these two highly mobile regions, a third (Gly 352 -Glu 359 ) is located at the beginning of the long C-terminal tail.…”

Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

“…Related glycosyltransferase matches were similarly identified by HHpred using Fks1 central domain regions from other fungi, as well as from distantly related orthologs found in plants and oomycetes (data not shown). The glycosyltransferase region homologous to S. cerevisiae Fks1 residues 1079 to 1158 includes the DXD motif which coordinates Mn 2ϩ interaction with the diphosphate of UDP-glucose substrate, and it is catalytically essential (3,14). In Fks1, the corresponding sequence is DAN (residues 1102 to 1104 [ Fig.…”

Polymethylated Polysaccharides from Mycobacterium Species Revisited

“…The crystal structure of the ortholog of Rv1208 in Mycobacterium avium spp. paratuberculosis (MAP2569c) has been solved (26,27), and diffraction-quality crystals of Rv1208 from M. tuberculosis were obtained. …”