A tethering mechanism for length control in a processive carbohydrate polymerization

Abstract

Carbohydrate polymers are the most abundant organic substances on earth. Their degrees of polymerization range from tens to thousands of units, yet polymerases generate the relevant lengths without the aid of a template. To gain insight into template-independent length control, we investigated how the mycobacterial galactofuranosyl-transferase GlfT2 mediates formation of the galactan, a polymer of galactofuranose residues that is an integral part of the cell wall. We show that isolated recombinant GlfT2 can catalyze the synthesis of polymers with degrees of polymerization that are commensurate with values observed in mycobacteria, indicating that length control by GlfT2 is intrinsic. Investigations using synthetic substrates reveal that GlfT2 is processive. The data indicate that GlfT2 controls length by using a substrate tether, which is distal from the site of elongation. The strength of interaction of that tether with the polymerase influences the length of the resultant polymer. Thus, our data identify a mechanism for length control by a template-independent polymerase.

Fig. 1.

Proposed role for GlfT2 in mycobacterial galactan biosynthesis. GlfT2 is hypothesized to mediate synthesis of the galactan polymer, which contains between 20 and 40 Galf residues (m = 10–20) connected by alternating β-Galf-(1→5)-Galf and β-Galf-(1→6)-Galf linkages. Elongation occurs by addition of Galf residues from UDP-Galf to the nonreducing end of a lipid-linked acceptor (compound 1). Synthetic acceptors 2-6 share features with the natural acceptor (compound 1). Rha, rhamnose.

Fig. 2.

GlfT2 alone can determine galactan length. The mass spectrum is shown from MALDI-TOF MS analysis of a 20-h incubation of a reaction mixture that contained His₆-GlfT2, UDP-Galf, and compound 5. Peaks that correspond to m/z values of [M + Na]⁺, in which M equals the mass of compound 5 plus n Galf residues, are labeled with the value of n. Unlabeled peaks have m/z values that correspond to the loss of the lipid moiety from elongation products, presumably because of fragmentation. Reaction products are observed from n = 3 to n = 27.

Fig. 3.

GlfT2 uses a processive mechanism for polymerization. Mass spectra are shown from MALDI-TOF MS analysis of time points of a reaction that contained His₆-GlfT2, UDP-Galf, and compound 5. Peaks that correspond to m/z values of [M + Na]⁺, in which M equals the mass of compound 5 plus n Galf residues, are labeled with n. At 2 min (≈60 turnovers), products are observed from n = 4–11. At 4 min (≈175 turnovers), products are observed from n = 3–15. At 8 min (≈440 turnovers), products are observed from n = 3–17. For all time points, we observed a peak of high intensity corresponding to the mass of unelongated acceptor (n = 0).

Fig. 4.

Proposed tethering model for GlfT2 (tan). (A) With acceptors that cannot occupy the lipid-anchoring site (e.g., compounds 2 or 3), only short oligomers are generated. (B) When GlfT2 interacts with acceptors that can bind to both sites (e.g., compound 5), longer oligomers are obtained. (C) When the polymer chain becomes long, polymer dissociation competes with further elongation.

Fig. 5.

Termination of polymerization depends on bivalent interactions of the substrate. Mass spectra from MALDI-TOF MS analysis of 20-h reactions with lower initial concentrations of compound 5 showed products with a greater degree of polymerization than those from reactions with higher initial substrate concentrations. In each spectrum, the peak that had the highest intensity of the group of high-molecular-weight peaks is labeled with n, which corresponds to [M + Na]⁺, where M equals the mass of compound 5 plus n Galf residues. The value of n increased with decreasing initial concentrations of compound 5.

Fig. 6.

Improved tethering leads to increased polymer lengths. The mass spectrum, from m/z = 5,000 to m/z = 9,000, is shown from a MALDI-TOF MS analysis of a 20-h incubation of a reaction mixture that contained His₆-GlfT2, UDP-Galf, and compound 6. Peaks that correspond to m/z values of [M + Na]⁺, in which M equals the mass of compound 6 plus n Galf residues, are labeled with the value of n. Unlabeled peaks have m/z values that correspond to the loss of the lipid moiety from elongation products, presumably because of fragmentation. Products are observed up to n = 48.