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 ␣-D-glucosamine-6-phosphate to ␣-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.

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…One group is clustered together with genes encoding the GTs identified by genetic and mass spec-trometry work as being involved in N-glycosylation and with genes whose roles in acetamido sugar biosynthesis have been demonstrated in vitro following expression and purification of the protein products in E. coli. Presently, this region encompasses mmp1076-mmp1094 (108,119,125). The second cluster is located in the region bounded by at least mmp0350-mmp0359 (S. Siu, J. Kelly, S. Logan, and K. F. Jarrell, unpublished data).…”

“…In addition to the genetic and mass spectrometry analysis of the M. maripaludis archaellin N-glycan, significant insight into the biosynthesis of the component sugars of the glycan have been provided by in vitro studies by Namboori and Graham showing that Methanococcales use a bacterial pathway to generate GlcNAc (108). The difficulties that were overcome in this study include the problem that key enzymes involved in GlcNAc biosynthesis cannot be identified simply by comparative sequence analysis since many belong to large protein families with diverse functions.…”

“…In addition, in vitro biochemical work using purified enzymes has further con-tributed to our understanding of the process in this group of Archaea (108)(109)(110).…”

“…Based on the results of these in vitro assays, the following sequence of events was proposed (108). Starting with Fru-6-P, an isomerizing glutamine-Fru-6-P transaminase, MMP1680 (GlmS), produces glucosamine-6-phosphate (GlcN-6-P).…”

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

“…The amino-terminal polyhistidine-tagged MMP0352 protein (His 10 -MMP0352) was heterologously expressed in E. coli BL21 (DE3) (pDG441) cells and purified by Ni 2+ -affinity chromatography using standard methods [17]. The molecular mass and purity of the protein was estimated by SDS-PAGE using the Laemmli buffer system and 12% total acrylamide.…”