Acetamido sugar biosynthesis in the Euryarchaea

Abstract

Archaea and eukaryotes share a dolichol phosphate-dependent system for protein N-glycosylation. In both domains, the acetamido sugar N-acetylglucosamine (GlcNAc) forms part of the core oligosaccharide. However, the archaeal Methanococcales produce GlcNAc using the bacterial biosynthetic pathway. Key enzymes in this pathway belong to large families of proteins with diverse functions; therefore, the archaeal enzymes could not be identified solely using comparative sequence analysis. Genes encoding acetamido sugar-biosynthetic proteins were identified in Methanococcus maripaludis using phylogenetic and gene cluster analyses. Proteins expressed in Escherichia coli were purified and assayed for the predicted activities. The MMP1680 protein encodes a universally conserved glucosamine-6-phosphate synthase. The MMP1077 phosphomutase converted alpha-D-glucosamine-6-phosphate to alpha-D-glucosamine-1-phosphate, although this protein is more closely related to archaeal pentose and glucose phosphomutases than to bacterial glucosamine phosphomutases. The thermostable MJ1101 protein catalyzed both the acetylation of glucosamine-1-phosphate and the uridylyltransferase reaction with UTP to produce UDP-GlcNAc. The MMP0705 protein catalyzed the C-2 epimerization of UDP-GlcNAc, and the MMP0706 protein used NAD(+) to oxidize UDP-N-acetylmannosamine, forming UDP-N-acetylmannosaminuronate (ManNAcA). These two proteins are similar to enzymes used for proteobacterial lipopolysaccharide biosynthesis and gram-positive bacterial capsule production, suggesting a common evolutionary origin and a widespread distribution of ManNAcA. UDP-GlcNAc and UDP-ManNAcA biosynthesis evolved early in the euryarchaeal lineage, because most of their genomes contain orthologs of the five genes characterized here. These UDP-acetamido sugars are predicted to be precursors for flagellin and S-layer protein modifications and for the biosynthesis of methanogenic coenzyme B.

FIG. 1.

Eukaryotes use the Leloir pathway to produce UDP-GlcNAc (22). The yeast Gfa1p protein is a d-Fru-6-P amidotransferase that produces GlcN-6-P. The Gna1p acetyltransferase produces N-acetylglucosamine 6-phosphate (GlcNAc-6-P), which can be converted to GlcNAc-1-P by the Agm1p (Pcm1p) phosphomutase. Finally, the Uap1p (Qri1p) uridylyltransferase produces UDP-GlcNAc. Yeast cells can also incorporate exogenous glucosamine, using separate acetyltransferase and kinase enzymes. Bacteria use the GlmS amidotransferase, the GlmM phosphomutase, and the bifunctional GlmU acetyltransferase/uridylyltransferase enzymes to produce UDP-GlcNAc (21). The WecB epimerase and the WecC dehydrogenase make UDP-ManNAcA for enterobacterial common-antigen biosynthesis (in some gram-negative proteobacteria) and capsule formation (in some gram-positive bacteria).

FIG. 2.

Biosynthesis of UDP-ManNAcA in the Methanococcales. The isomerizing glutamine-Fru-6-P transaminase (GlmS; EC 2.6.1.16; MJ1420 or MMP1680) catalyzes the isomerization of Fru-6-P and its transamination from the l-glutamine amide, producing GlcN-6-P. Phosphoglucosamine mutase (GlmM; EC 5.4.2.10; MJ1100 or MMP1077) catalyzes the transfer of phosphate from GlcN-6-P to the anomeric position, forming α-d-glucosamine-1-phosphate (GlcN-1-P). The bifunctional GlcN-1-P uridylyltransferase/acetyltransferase (GlmU; EC 2.7.7.23/2.3.1.57; MJ1101 or MMP1076) catalyzes the acetylation of the 2-amino group using acetyl-CoA and the transfer of GlcNAc-1-P to UTP, releasing pyrophosphate and UDP-GlcNAc. The UDP-GlcNAc 2-epimerase (WecB; EC 5.1.3.14; MJ1504 or MMP0705) catalyzes the isomerization of UDP-GlcNAc to produce UDP-ManNAc. Finally, the UDP-ManNAc 6-dehydrogenase (WecC; EC 1.1.1.-; MJ0468 or MMP0706) catalyzes the four-electron oxidation of UDP-ManNAc, reducing NAD⁺ and releasing UDP-ManNAcA.

FIG. 3.

Purified proteins analyzed by SDS-PAGE on 12% acrylamide gels and stained with Coomassie blue. Lane 1, 10 μg of MMP1680; lane 2, 8.5 μg of His₁₀-MMP1077; lane 3, 8 μg of His₁₀-MJ1101; lane 4, protein markers corresponding to the indicated molecular masses; lane 5, 8 μg of MMP0705-His₆; lane 6, 10 μg of His₆-MMP0706; lane 7, protein markers.

FIG. 4.

The MMP1680 protein catalyzes the isomerization and transamidation of Fru-6-P to form GlcN-6-P. Amines from reactions containing purified protein, l-glutamine (Gln), and Fru-6-P were derivatized to form fluorescent 1-cyanobenz[f]-isoindole derivatives of GlcN-6-P, Gln, and glutamate (Glu) products, which were separated by reversed-phase HPLC. Reactions without MMP1680 protein showed only a Gln peak, but no GlcN-6-P or Glu products. The experimental conditions are described in the text.

FIG. 5.

Chromatogram showing the separation of nucleotides by HPLC on a CarboPac PA1 column. (A) Reactions containing His₁₀-MMP1077, His₁₀-MJ1101, UTP, Ac-CoA, GlcN-6-P, and pyrophosphatase produced UDP-GlcNAc and CoA. No UDP-GlcNAc or CoA was observed in reactions without His₁₀-MMP1077. (B) The MMP0705-His₆ protein catalyzed the epimerization of UDP-GlcNAc to form UDP-ManNAc (not resolved) and UDP, an intermediate in the epimerase reaction formed by the enzymatic cleavage of UDP sugars. No UDP or early-eluting product was detected in control reactions without MMP0705-His₆ protein. (C) Reactions containing MMP0705-His₆, His₆-MMP0706, UDP-GlcNAc, and NAD⁺ produced UDP-ManNAcA and two equivalents of NADH. Reactions without the His₆-MMP0706 protein did not produce UDP-ManNAcA or NADH. The minor unlabeled peaks in the chromatograms were detected in both enzymatic and control reactions. The full experimental conditions are described in the text.

FIG. 6.

A coupled assay measured the UDP-GlcNAc 2-epimerase activity of MMP0705. (A) Reaction progress curves show activities for coupled epimerase and dehydrogenase reactions containing 3 mM UDP-GlcNAc (filled circles) and 1.5 mM UDP-GlcNAc (open circles). Control reactions without MMP0705 enzyme (filled squares) or without UDP-GlcNAc (open squares) show background levels of activity. The reaction mixtures contained 3 to 5 mM NAD⁺, 0.4 μg MMP0705-His₆, and 150 μg His₆-MMP0706 with buffer salts, as described in the text. The reduction of NAD⁺ to NADH was monitored by following the increase in absorbance at 340 nm. (B) Hill plot of the rate of MMP0705-catalyzed epimerization of UDP-GlcNAc. Initial rate data were fitted to the Hill equation by nonlinear regression. The Hill coefficient was 2.7, the K_M for UDP-GlcNAc was 0.36 mM, and the k_cat was 3.4 s⁻¹.