Mechanistic insights into the retaining glucosyl-3-phosphoglycerate synthase from mycobacteria

Abstract

Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.

FIGURE 1.

MGLP biosynthesis in mycobacteria. A, glucosyl-3-phosphoglycerate biosynthesis in mycobacteria. GpgS transfers a Glcp residue from UDP-Glcp to the 2-position of 3-phosphoglycerate to form glucosyl-3-phosphoglycerate. The reaction occurs with retention of the anomeric configuration of the sugar donor. B, MGLPs chemical structure. The MGLPs from Mycobacterium bovis BCG are composed of 10 α-(1→4)-linked 6-O-methylglucosyl residues with a nonreducing end made of the tetrasaccharide 3-O-methyl-d-Glcp-(α-(1→4)-d-Glcp)₃-α-(1→. The tetrasaccharide →4)-(α-(1→4)-d-Glcp)₃-α-(1→6)-d-Glcp-α-(1→ linked to position 2 of d-glyceric acid constitutes the reducing end of the molecule) (66). Position 3 of the second and that of fourth α-d-Glcp residues (closest to the reducing end) are substituted by single α-d-Glcp residues. R1, R2, and R3 are acyl groups: R1, acetate, propionate, or isobutyrate; R2, octanoate; and R3, succinate. MGLPs occur as a mixture of four main components that differ in their content of esterified succinate. The names of the genes thought to be involved in the different steps of their elongation and modifications are shown (32, 67). Acylation and methylation are thought to occur concurrently; the precise stage at which the two β-(1→3)-linked Glc residues are attached is not known, but the definition of early MGLP precursors suggests that they are added early during the elongation process.

FIGURE 2.

Role of metal ions in MtGpgS enzymatic activity. The MtGpgS activity was measured by incubating the recombinant enzyme in the presence of UDP-d-[U-¹⁴C]Glc, d-3-phosphoglyceric acid (Sigma), and a broad range of metal ion salts as follows: 1st lane, no enzyme added; 2nd lane, no metal ion added; 3rd lane, MgCl₂; 4th lane, CoCl₂; 5th lane, CaCl₂; 6th lane, ZnCl₂; 7th lane, FeCl₂; 8th lane, CuCl₂; 9th lane, MnCl₂; 10th lane, MgCl₂ and EDTA (for details see “Experimental Procedures”).

FIGURE 3.

Two different conformations, L_A and L_I, for the catalytic loop. A, overall structure of MtGpgS in complex with Mn²⁺·UDP·PGA. The core of the protein consists of an eight-stranded continuous β-sheet with topology β4-β3-β2-β5-β7-β8-β1 (β7 is antiparallel) flanked by three α-helices on either side. The sugar donor-binding domain includes residues 45–138 (orange), whereas the acceptor-binding domain is made of residues 139–262 (yellow). A large C-terminal extension (residues 262–323) containing an α-helix and a two-stranded antiparallel twisted β-sheet (β11–β12), are rich in aromatic residues and involved in protein dimerization (purple). The second monomer is shown in pink. B, structural comparison of the catalytic loop as observed in the ternary MtGpgS·Mn²⁺·UDP·PGA (red) complex and in its inactive conformation L_I in the apo-form (blue). C, structural comparison of the catalytic loop as observed in the ternary MtGpgS·Mn²⁺·UDP·PGA (red) complex and in its active conformation L_A in the apo-form (pink). D, structural comparison of selected region of mycobacterial GpgSs. The catalytic loop as observed in binary and ternary complexes of MaGpgS and MtGpgS are shown in red. The active conformation L_A in the apo-form is shown in pink. The inactive conformation L_I in the apo-form of MtGpgS is shown in blue.

FIGURE 4.

Donor recognition site, two conformations for the α- and β-phosphates. A, structural comparison of a selected region of the binary MaGpgS·Mn²⁺·UDP-Glc (41) and ternary MtGpgS·Mn²⁺·UDP·PGA (this study) complexes. The catalytic loop is shown in red. B, active site of MgS from R. marinus in the same orientation as GpgS. C and D, schematic representation showing MtGpgS and MgS, respectively, associated to their sugar donor and acceptor substrates.

FIGURE 5.

ITC measurements of GpgS-ligand interactions. A, binding isotherms for the binding of UDP-Glc (▾), UDP (■), UMP (□), and uridine (URI, △) to MtGpgS protein at 15 °C. The upper panel shows the raw heat signal for successive injections of a ligand solution into the protein solution. The lower panel shows the integrated heats of injections corrected for the heat of dilution and normalized to ligand concentration. Solid lines correspond to a best fit of the isotherm to the GpgS P0↔ P1 conformational equilibrium model for UDP-Glc and UDP (cf. text) and to a bimolecular binding model for URI and UMP. Inset, enthalpies and free energies of binding of UDP (●, ○) and UDP-Glc (▴, △) to the P₁ conformation as a function of temperature. Solid lines correspond to linear fits of the data. B, binding isotherms for the binding of PGA to free MtGpgS (●) and to MtGpgS complexes formed with UDP (■), UMP (▴), and URI (□) at 15 °C. Inset, same as the inset in A for the binding of PGA to the UDP complex (■, □). B, MtGpgS was first titrated with a nucleotide as described in A, and the protein·nucleotide complex formed was then titrated with PGA. Thermodynamic data are reported in Table 4.

FIGURE 6.

Overall conformational changes in MtGpgS. A, trypsin cleavage of MtGpgS preincubated with different ligands. B and C, AUC studies of MtGpgS and of MtGpgS·ligand complexes. MtGpgS (○) was incubated alone or with equimolar amounts of UDP-Glc (♢), UDP (■), UMP (▴), URI (+), PGA (△), and UDP-PGA (♦) prior to sedimentation velocity experiments. The resulting integral distribution of S (corrected for water at 20 °C (s_20,w)) is shown.