Archaeal Enzyme Research Articles

Identification of Amino Acids in the N-terminal Domain of Atypical Methanogenic-type Seryl-tRNA Synthetase Critical for tRNA Recognition

Seryl-tRNA synthetase (SerRS) from methanogenic archaeon Methanosarcina barkeri, contains an idiosyncratic N-terminal domain, composed of an antiparallel beta-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. It is very different from the coiled-coil tRNA binding domain in bacterial-type SerRS. Because the crystal structure of the methanogenic-type SerRSxtRNA complex has not been obtained, a docking model was produced, which indicated that highly conserved helices H2 and H3 of the N-terminal domain may be important for recognition of the extra arm of tRNA(Ser). Based on structural information and the docking model, we have mutated various positions within the N-terminal region and probed their involvement in tRNA binding and serylation. Total loss of activity and inability of the R76A variant to form the complex with cognate tRNA identifies Arg(76) located in helix H2 as a crucial tRNA-interacting residue. Alteration of Lys(79) positioned in helix H2 and Arg(94) in the loop between helix H2 and beta-strand A4 have a pronounced effect on SerRSxtRNA(Ser) complex formation and dissociation constants (K(D)) determined by surface plasmon resonance. The replacement of residues Arg(38) (located in the loop between helix H1 and beta-strand A2), Lys(141) and Asn(142) (from H3), and Arg(143) (between H3 and H4) moderately affect both the serylation activity and the K(D) values. Furthermore, we have obtained a striking correlation between these results and in vivo effects of these mutations by quantifying the efficiency of suppression of bacterial amber mutations, after coexpression of the genes for M. barkeri suppressor tRNA(Ser) and a set of mMbSerRS variants in Escherichia coli.

Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine N-methyltransferase from Methanohalophilus portucalensis

To overcome the extracellular salt stress, Methanohalophilus portucalensis FDF1(T) synthesizes the compatible solute betaine through the methylation of glycine, sarcosine, and N,N-dimethylglycine. S-adenosylmethionine (AdoMet) is the methyl donor. The enzyme sarcosine dimethylglycine N-methyltransferase (SDMT) of M. portucalensis, that catalyzes the formation of N,N-dimethylglycine and glycine betaine, has been purified and characterized. SDMT, a monomer of 33 kDa with a pI at 5.03, has a narrow substrate specificity limited to using only sarcosine and dimethylglycine as substrates for the methyl transferase reaction. The K(m) values for sarcosine and AdoMet were 2.29 and 0.21 mM, respectively, with a V(max) of 0.83 micromol/mg-min (k(cat) value of 0.44 s(-1)). The K(m) values for dimethylglycine and AdoMet were 3.76 and 0.59 mM, respectively, with a V(max) of 4.88 micromol/mg-min (k(cat) of 2.68 s(-1)). A high concentration of the end product betaine (2.0 M) did not affect the SMT activity, but it slightly inhibited the DMT activity. Both activities were also not affected by potassium or sodium ions in concentrations of 200-1,000 mM. We compared this novel archaeal SDMT enzyme to other similar bacterial transferases as well as to the glycine sarcosine dimethylglycine methyltransferase found also in M. portucalensis.

Novel Domain Arrangement in the Crystal Structure of a Truncated Acetyl-CoA Synthase from Moorella thermoacetica,

Ni-dependent acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) constitute the central enzyme complex of the Wood-Ljungdahl pathway of acetyl-CoA formation. The crystal structure of a recombinant bacterial ACS lacking the N-terminal domain that interacts with CODH shows a large reorganization of the remaining two globular domains, producing a narrow cleft of suitable size, shape, and nature to bind CoA. Sequence comparisons with homologous archaeal enzymes that naturally lack the N-terminal domain show that many amino acids lining this cleft are conserved. Besides the typical [4Fe-4S] center, the A-cluster contains only one proximal metal ion that, according to anomalous scattering data, is most likely Cu or Zn. Incorporation of a functional Ni(2)Fe(4)S(4) A-cluster would require only minor structural rearrangements. Using available structures, a plausible model of the interaction between CODH and the smaller ACS in archaeal multienzyme complexes is presented, along with a discussion of evolutionary relationships of the archaeal and bacterial enzymes.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

DNA Ligases: Progress and Prospects

DNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates. DNA ligases are essential guardians of genomic integrity, and ligase dysfunction underlies human genetic disease syndromes. Crystal structures of DNA ligases bound to nucleotide and nucleic acid substrates have illuminated how ligase reaction chemistry is catalyzed, how ligases recognize damaged DNA ends, and how protein domain movements and active-site remodeling are used to choreograph the end-joining pathway. Although a shared feature of DNA ligases is their envelopment of the nicked duplex as a C-shaped protein clamp, they accomplish this feat by using remarkably different accessory structural modules and domain topologies. As structural, biochemical, and phylogenetic insights coalesce, we can expect advances on several fronts, including (i) pharmacological targeting of ligases for antibacterial and anticancer therapies and (ii) the discovery and design of new strand-sealing enzymes with unique substrate specificities.

Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA

Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 A of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

Effect of mutagenesis at the region upstream from the G(Q/E) motif of three types of geranylgeranyl diphosphate synthase on product chain-length

(All-E) geranylgeranyl diphosphate synthases have been classified into three types based on the characteristic sequences around the first aspartate rich motif, which is highly conserved among the enzymes. In type I geranylgeranyl diphosphate synthases, which consist of archaeal enzymes, a bulky amino acid residue at the 5th position upstream from the motif plays a main role in the product determination, by blocking further elongation of prenyl chain as the bottom of the reaction cavity. On the other hand, type III geranylgeranyl diphosphate synthases, which consist of the enzymes from eukaryotes except for plants, use a bulky amino acid residue at the 2nd position upstream from the conserved G(Q/E) motif for product chain-length determination. Thus we introduced mutations into the region upstream from the G(Q/E) motif of geranylgeranyl diphosphate synthases of the three different types to confirm the importance of the region for the product chain-length determination. The results of the mutational analyses indicated that not only the 2nd but also the 3rd position upstream from the G(Q/E) motif is involved in the product chain-length determination mechanism in types I and III geranylgeranyl diphosphate synthases, while the amino acid substitution in this region did not affect the chain-length of the products of type II geranylgeranyl diphosphate synthase, which consist of the enzymes from bacteria and plants. The region upstream from the G(Q/E) motif possibly contributes to the product determination in the wide range of geranylgeranyl diphosphate synthases, as well as that around the first aspartate rich motif.

Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure-function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the alpha subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the DeltaH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro

The general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N-terminal zinc ribbon and some highly conserved residues in the C-terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The ΔP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the ΔP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild-type P in transcription reactions. The conserved zinc ribbon in the N-terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17-amino-acid C-terminal peptide is sufficient to support all basic RNA polymerase functions in vitro.

Phosphomannose isomerase/GDP-mannose pyrophosphorylase from Pyrococcus furiosus: a thermostable biocatalyst for the synthesis of guanidinediphosphate-activated and mannose-containing sugar nucleotides

Herein we present an analysis of the chemical function of a recombinant bifunctional phosphomannose isomerase/GDP-mannose pyrophosphorylase (manC) from Pyrococcus furiosus DSM 3638 and its use in the synthesis of guanidinediphospho-hexoses and a range of nucleotidediphospho-mannoses. This enzyme is unusually promiscuous in both its nucleotide triphosphate (NTP) and sugar-1-phosphate acceptance. It accepts all five naturally occurring NTPs (ATP, CTP, GTP, dTTP and UTP) and a range of sugar-1-phosphates (glucose-, mannose-, galactose-, glucosamine-, N-acetylglucosamine- and fucose-1-phosphate). A truncated GDP-mannose pyrophosphorylase domain of the whole length enzyme showed almost 100-fold less sugar nucleotidyltransferase activity with only GTP and mannose 1-phosphate as substrates. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme make it an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides that are challenging to make by chemical means alone.

Structure–function relationships in Escherichia coli adenylate cyclase

Class I adenylate cyclases are found in gamma- and delta-proteobacteria. They play central roles in processes such as catabolite repression in Escherichia coli or development of full virulence in pathogens such as Yersinia enterocolitica and Vibrio vulnificus. The catalytic domain (residues 2-446) of the adenylate cyclase of E. coli was overexpressed and purified. It displayed a V(max) of 665 nmol of cAMP x mg(-1) x min(-1) and a K(m) of 270 microM. Titration of the metal cofactor Mg(2+) against the substrate ATP showed a requirement for free metal ions in addition to the MgATP complex, suggesting a two-metal-ion mechanism as is known for class II and class III adenylate cyclases. Twelve residues which are essential for catalysis were identified by mutagenesis of a total of 20 polar residues conserved in all class I adenylate cyclases. Five essential residues (Ser(103), Ser(113), Asp(114), Asp(116) and Trp(118)) were part of a region which is found in all members of the large DNA polymerase beta-like nucleotidyltransferase superfamily. Alignment of the E. coli adenylate cyclase with the crystal structure of a distant member of the superfamily, archaeal tRNA CCA-adding enzyme, suggested that Asp(114) and Asp(116) are the metal-cofactor-ion-binding residues. The S103A mutant had a 17-fold higher K(m) than wild-type, demonstrating its important role in substrate binding. In comparison with the tRNA CCA-adding enzyme, Ser(103) of the E. coli adenylate cyclase apparently binds the gamma-phosphate group of ATP. Consistent with this function, the S103A mutation caused a marked reduction of discrimination between ATP- and ADP- or AMP-derived inhibitors.

Structural Insight into the Bifunctional Mechanism of the Glycogen-debranching Enzyme TreX from the Archaeon Sulfolobus solfataricus

TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase and alpha-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp363, occupying subsites -1 to -3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the "flexible loop" (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in alpha-1,4-transferase activity and a reduced level of alpha-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99-97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the alpha-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.

Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase

Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the Gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1 Å resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.

2’-5’ Oligoadenylate synthetase shares active site architecture with the archaeal CCA-adding enzyme

The 2'-5' oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognise virally produced dsRNA and initiate an RNA destabilisation within the infected cell. We compared the structure of OAS to that of poly adenosine polymerase (PAP) and the class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA). This comparison revealed a strong structural homology between the three enzyme families. In particular, the active sites of OAS and CCA class I enzymes are highly conserved. We conducted an extensive mutagenesis of amino acid residues within the putative active site in OAS, thereby identifying enzymatically important residues and confirming the common active site architecture for OAS and the AfCCA. Our findings also have profound implications for our understanding of the evolutionary origin of the OAS enzymes and suggest that the OAS proteins diverged from a common 3'-specific ancestor at the beginning of metazoan evolution.

Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.

Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

The modified flavin coenzyme F(420) is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F(420)-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F(420) for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F(420) and citrate at resolutions of 1.90 and 1.95 A(,) respectively. The structure reveals a highly specific F(420) binding mode, which is shared with several other F(420)-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F(420) and is shown to be a competitive inhibitor (IC(50) 43 microm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F(420) and helps promote a butterfly bend conformation that facilitates F(420) reduction and catalysis.

Structural evolution of multisubunit RNA polymerases

Evolutionarily related multisubunit RNA polymerases (RNAPs) facilitate gene transcription throughout the three domains of life. During the past seven years an increasing number of bacterial and eukaryotic RNAP structures have been solved; however, the archaeal enzyme remained elusive. Two reports from the Murakami and Cramer laboratories have now filled this gap in our knowledge and enable us to hypothesize about the evolution of the structure and function of RNAPs.

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding α-helix of archaeal CCA-adding enzymes

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.

Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O -phosphoseryl-tRNA Sec kinase

Selenocysteine (Sec) biosynthesis in archaea and eukaryotes requires three steps: serylation of tRNASec by seryl-tRNA synthetase (SerRS), phosphorylation of Ser-tRNASec by O-phosphoseryl-tRNASec kinase (PSTK), and conversion of O-phosphoseryl-tRNASec (Sep-tRNASec) by Sep-tRNA:Sec-tRNA synthase (SepSecS) to Sec-tRNASec. Although SerRS recognizes both tRNASec and tRNASer species, PSTK must discriminate Ser-tRNASec from Ser-tRNASer. Based on a comparison of the sequences and secondary structures of archaeal tRNASec and tRNASer, we introduced mutations into Methanococcus maripaludis tRNASec to investigate how Methanocaldococcus jannaschii PSTK distinguishes tRNASec from tRNASer. Unlike eukaryotic PSTK, the archaeal enzyme was found to recognize the acceptor stem rather than the length and secondary structure of the D-stem. While the D-arm and T-loop provide minor identity elements, the acceptor stem base pairs G2-C71 and C3-G70 in tRNASec were crucial for discrimination from tRNASer. Furthermore, the A5-U68 base pair in tRNASer has some antideterminant properties for PSTK. Transplantation of these identity elements into the tRNASerUGA scaffold resulted in phosphorylation of the chimeric Ser-tRNA. The chimera was able to stimulate the ATPase activity of PSTK albeit at a lower level than tRNASec, whereas tRNASer did not. Additionally, the seryl moiety of Ser-tRNASec is not required for enzyme recognition, as PSTK efficiently phosphorylated Thr-tRNASec.

Acetamido Sugar Biosynthesis in the Euryarchaea

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.