Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions

Abstract

Asparagine-linked protein glycosylation is essential for the virulence of the human gut mucosal pathogen Campylobacter jejuni . The heptasaccharide that is transferred to proteins is biosynthesized via the glycosyltransferase-catalyzed addition of sugar units to an undecaprenyl diphosphate-linked carrier. Genetic studies on the heptasaccharide assembly enzymes have shown that PglH, which transfers three terminal N-acetyl-galactosamine (GalNAc) residues to the carrier polyisoprene, is essential for chick colonization by C. jejuni . While it is now clear that PglH catalyzes multiple transfer reactions, the mechanism whereby the reactions cease after the addition of just three GalNAc residues has yet to be understood. To address this issue, a series of mechanistic biochemical studies was conducted with purified native PglH. This enzyme was found to follow a processive mechanism under initial rate conditions; however, product inhibition and product accumulation led to PglH release of intermediate products prior to complete conversion to the native ultimate product. Point mutations of an essential EX(7)E sequence motif were used to demonstrate that a single active site was responsible for all three transferase reactions, and a homology model with the mannosyltransferase PimA, from Mycobacteria smegmatis , establishes the requirement of the EX(7)E motif in catalysis. Finally, increased binding affinity with increasing glycan size is proposed to provide PglH with a counting mechanism that does not allow the transfer of more than three GalNAc residues. These results provide important mechanistic insights into the function of the glycosyl transfer polymerase that is related to the virulence of C. jejuni .

Figure 1

Campylobacter jejuni polyisoprene-linked oligosaccharide biosynthesis. It is important to note that the stereochemistry of the bacterial undecaprenol double bonds are 2-trans to 8-cis rather than the 3-trans to 7-cis shown for the plant derived undecaprenol used here.

Figure 2

Potential mechanisms for the PglH reaction. a) Block transfer of a tri-GalNAc moiety to H₀, b) Processive mechanism in which intermediates do not dissociate from the enzyme, c) Dissociative mechanism in which intermediates freely dissociate and re-associate as substrates.

Figure 3

PglH utilizes the highly conserved EX₇E motif flanked by residues E265 and E273 for catalysis of all reactions. Reactions containing the indicated PglH mutants (20 nM) were prepared and the amount of product determined for an overnight reaction with H₀ (68 nM) and UDP-[³H]GalNAc (20 Ci/mmol; 250 nM).

Figure 4

The concentration of substrate determines the distribution of products and intermediates in a PglH reaction. PglH reactions were prepared with either a) 4.3 μM Und-PP-Bac2,4diNAc-[³H]GalNAc-GalNAc (H₀) and 0.25 μM UDP-[³H]GalNAc or b) 0.26 μM Und-PP-Bac2,4diNAc-[³H]GalNAc-GalNAc (H₀) and 0.25 μM UDP-[³H]GalNAc. Each column represents the amount of radioactivity in a 1 mL fraction at the corresponding time corrected for the number of [³H]GalNAc units incorporated into each. Note that with high polyisoprene-linked substrate relative to UDP-[³H]GalNAc the majority of the product is the first intermediate (H₁), and when the substrates are approximately equimolar the major product is the final product (H₃). The identity of H₁and H₂ were based on consistent normal phase HPLC retention times of the tritium-labeled PglH products and quantitative conversion of the isolated intermediate to H₃ upon further incubation with PglH.

Figure 5

A distribution of products and intermediates are formed in the PglH reaction. PglH reactions were prepared with 28 μM UDP-GalNAc and 4 μM Und-PP-Bac2,4diNAc-(GalNAc)₂ (H₀) and aliquots were extracted at the given time points then analyzed by NP-HPLC. a) The quantity of each intermediate was calculated based on their distribution as analyzed by NP-HPLC and the total counts in the reaction. Product concentration is provided at the indicated time points for total (closed circles), H₁ (open diamond), H₂ (closed squares) and H₃ (open triangle). b) The percentage of the total product was calculated for each time point and plotted H₁ (open diamond), H₂ (closed squares) and H₃ (open triangle). Note that H₂ stays at the same percentage throughout the reaction and that H₁ and H₂ levels sharply increase initially, but then stabilize as H₃ is the major product. Also, note that the lines are drawn for illustrative purposes only.

Figure 6

PglH is initially a processive enzyme that builds up product to a limit then intermediates form and dissociate. PglH reactions were prepared with 3 μM Und-PP-Bac2,4diNAc-(GalNAc)₂ (H₀) and 0.25 μM UDP-GalNAc. Reactions were quenched and extracted after 8 minutes (black), 32 minutes (gray) and overnight (white) then analyzed by normal phase HPLC. Each bar represents the total radioactivity associated with 1 mL fractions at the given time points. Radioactivity was corrected for the number of GalNAc units incorporated into each molecule. Note that the quantity of final product is high relative to the others initially, but at the end of the reaction the product is mostly the first intermediate.

Figure 7

PglH follows a sequential ordered path of substrate addition. PglH reactions were prepared with variable H₀ alone (closed diamonds) or with a) 94 nM H₃, (open triangles) or b) 10 μM UDP (open triangles) and the initial rates were determined then analyzed by Hanes-Woolf plots. Note that the H₃ product inhibition and H₀ lines are parallel indicating competitive inhibition and that the UDP product inhibition lines intersect at the x-axis suggesting noncompetitive inhibition.

Figure 8

PglH intermediates can compete with the natural substrate of the enzyme. Tritium-labeled Und-PP-Bac2,4diNAc-(GalNAc)₂ (H₀) and [¹⁴C]-labeled Und-PP-Bac2,4diNAc-(GalNAc)₃ (H₁) and Und-PP-Bac2,4diNAc-(GalNAc)₄ (H₂) were enzymatically synthesized then mixed with PglH. H₁ or H₂ were equimolar with H₀ (75 nM). The reactions were stopped after 2.5 minutes with H₁ or 20 minutes with H₂ and the distribution of products was analyzed by NP-HPLC. Each bar represents the number of counts detected in 1 mL fractions at the given time and the [¹⁴C] label is associated with the right-hand y-axis while tritium is associated with the left-hand y-axis. Overlap between the [³H] and [¹⁴C] are shown in gray. a) [³H]-labeled H₀ (black) and [¹⁴C]-labeled H₁ (white) b) [³H] labeled H₀ (black) and [¹⁴C] labeled H₂ (white) NP-HPLC traces. Note that the distribution of products is the same with the [¹⁴C] and [³H]-labeled substrates.