Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases.

The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on sequence similarity is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungal genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases.

Small-Molecule Acetylation by GCN5-Related N-Acetyltransferases in Bacteria.

Acetylation is a conserved modification used to regulate a variety of cellular pathways, such as gene expression, protein synthesis, detoxification, and virulence. Acetyltransferase enzymes transfer an acetyl moiety, usually from acetyl coenzyme A (AcCoA), onto a target substrate, thereby modulating activity or stability. Members of the GCN5- N -acetyltransferase (GNAT) protein superfamily are found in all domains of life and are characterized by a core structural domain architecture. These enzymes can modify primary amines of small molecules or of lysyl residues of proteins. From the initial discovery of antibiotic acetylation, GNATs have been shown to modify a myriad of small-molecule substrates, including tRNAs, polyamines, cell wall components, and other toxins. This review focuses on the literature on small-molecule substrates of GNATs in bacteria, including structural examples, to understand ligand binding and catalysis. Understanding the plethora and versatility of substrates helps frame the role of acetylation within the larger context of bacterial cellular physiology.

Emergence of the Novel Aminoglycoside Acetyltransferase Variant aac(6')-Ib-D179Y and Acquisition of Colistin Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae Due to a Disrupting Mutation in the DNA Repair Enzyme MutS.

Amikacin and colistin are effective against carbapenem-resistant Klebsiella pneumoniae In 2017, we successively isolated three carbapenem-resistant K. pneumoniae isolates (ST967) from a patient with chronic renal failure in Japan. The first (SMKP01, sputum, day 0) and second (SMKP02, blood, day 14) strains were resistant to most antimicrobials tested but still susceptible to amikacin (MICs of 4 and 0.5 mg/liter, respectively) and colistin (MIC of 0.5 mg/liter for both). The third strain (SMKP03, blood, day 51) was not susceptible to amikacin (MIC, 32 mg/liter), and its MIC for colistin varied (0.5 to 8 mg/liter). Whole-genome sequencing of SMKP01 revealed that 17 of 20 antimicrobial resistance genes, including qnrB91 (a novel qnrB2 variant) and aac(6')-Ib-cr, were located on an 86.9-kb IncFII-IncQ plasmid. The qnrB91 conferred greater fluoroquinolone resistance than qnrB2 SMKP03 aac(6')-Ib-cr that possessed a gene mutation that resulted in an R102W substitution, namely, aac(6')-Ib-D179Y, made a greater contribution to amikacin resistance than did aac(6')-Ib-cr SMKP03 harbored a nonsense mutation in mutS, which encodes a DNA repair enzyme. Introduction of this mutation into SMKP01 (SMKP01mutSA307T) resulted in a dramatic increase (>58-fold) in the frequency of spontaneous amikacin-resistant mutants relative to SMKP01, and the substantial mutants possessed aac(6')-Ib-D179Y SMKP01mutSA307T exhibited an unstable MIC for colistin (0.5 to 8 mg/liter). The results demonstrate that a disruptive mutation in MutS, arising during the clinical course of an infection, created a platform for the acquisition of amikacin nonsusceptibility and colistin heteroresistance in multidrug-resistant K. pneumoniae, mediated by the elevated frequency of spontaneous mutations.IMPORTANCE The emergence of multidrug resistance in pathogens such as Klebsiella pneumoniae is of great clinical concern. Antimicrobial resistance sometimes arises during the course of an infection. Although many studies have reported the emergence of antimicrobial resistance and novel antimicrobial resistance genes in the clinical isolates, the identity of the bacterial factor(s) that generate this emergence is still unclear. We report that a disruptive mutation in MutS, arising during the clinical course of an infection, created a context for the acquisition of colistin resistance and the emergence of a novel variant of the amikacin resistance gene in multidrug-resistant K. pneumoniae via an increase in the frequency of spontaneous mutation. This observation is important for understanding how K. pneumoniae develops multidrug resistance during infection and could potentially lead to new antimicrobial treatments for high-risk pathological microbes.

Comprehensive analysis of protein acetyltransferases of human pathogen Mycobacterium tuberculosis.

Tuberculosis (TB), a leading infectious disease caused by Mycobacterium tuberculosis strain, takes four human lives every minute globally. Paucity of knowledge on M. tuberculosis virulence and antibiotic resistance is the major challenge for tuberculosis control. We have identified 47 acetyltransferases in the M. tuberculosis, which use diverse substrates including antibiotic, amino acids, and other chemical molecules. Through comparative analysis of the protein file of the virulent M. tuberculosis H37Rv strain and the avirulent M. tuberculosis H37Ra strain, we identified one acetyltransferase that shows significant variations with N-terminal deletion, possibly influencing its physicochemical properties. We also found that one acetyltransferase has three types of post-translation modifications (lysine acetylation, succinylation, and glutarylation). The genome context analysis showed that many acetyltransferases with their neighboring genes belong to one operon. By data mining from published transcriptional profiles of M. tuberculosis exposed to diverse treatments, we revealed that several acetyltransferases may be functional during M. tuberculosis infection. Insights obtained from the present study can potentially provide clues for developing novel TB therapeutic interventions.

Contrasting evolutionary patterns of multiple loci uncover new aspects in the genome origin and evolutionary history of Leymus (Triticeae; Poaceae).

Leymus Hochst. (Triticeae: Poaceae), a group of allopolyploid species with the NsXm genomes, is a perennial genus with diversity in morphology, cytology, ecology, and distribution in the Triticeae. To investigate the genome origin and evolutionary history of Leymus, three unlinked low-copy nuclear genes (Acc1, Pgk1, and GBSSI) and three chloroplast regions (trnL-F, matK, and rbcL) of 32 Leymus species were analyzed with those of 36 diploid species representing 18 basic genomes in the Triticeae. The phylogenetic relationships were reconstructed using Bayesian inference, Maximum parsimony, and NeighborNet methods. A time-calibrated phylogeny was generated to estimate the evolutionary history of Leymus. The results suggest that reticulate evolution has occurred in Leymus species, with several distinct progenitors contributing to the Leymus. The molecular data in resolution of the Xm-genome lineage resulted in two apparently contradictory results, with one placing the Xm-genome lineage as closely related to the P/F genome and the other splitting the Xm-genome lineage as sister to the Ns-genome donor. Our results suggested that (1) the Ns genome of Leymus was donated by Psathyrostachys, and additional Ns-containing alleles may be introgressed into some Leymus polyploids by recurrent hybridization; (2) The phylogenetic incongruence regarding the resolution of the Xm-genome lineage suggested that the Xm genome of Leymus was closely related to the P genome of Agropyron; (3) Both Ns- and Xm-genome lineages served as the maternal donor during the speciation of Leymus species; (4) The Pseudoroegneria, Lophopyrum and Australopyrum genomes contributed to some Leymus species.

Functional characterization and differential nutritional regulation of putative Elovl5 and Elovl4 elongases in large yellow croaker (Larimichthys crocea).

In the present study, two elongases, Elovl4 and Elovl5, were functionally characterized and their transcriptional regulation in response to n-3 LC-PUFA administration were investigated in vivo and in vitro. We previously described the molecular characterization of croaker elovl5. Here, we report the full-length cDNA sequence of croaker elovl4, which contained 1794 bp (excluding the polyA tail), including 909 bp of coding region that encoded a polypeptide of 302 amino acids possessing all the characteristic features of Elovl proteins. Functional studies showed that croaker Elovl5, displayed high elongation activity towards C18 and C20 PUFA, with only low activity towards C22 PUFA. In contrast, croaker Elovl4 could effectively convert both C20 and C22 PUFA to longer polyenoic products up to C34. n-3 LC-PUFA suppressed transcription of the two elongase genes, as well as srebp-1 and lxrα, major regulators of hepatic lipid metabolism. The results of dual-luciferase reporter assays and in vitro studies both indicated that the transcriptions of elovl5 and elovl4 elongases could be regulated by Lxrα. Moreover, Lxrα could mediate the transcription of elovl4 directly or indirectly through regulating the transcription of srebp-1. The above findings contribute further insight and understanding of the mechanisms regulating LC-PUFA biosynthesis in marine fish species.

Crystal structure of Pseudomonas aeruginosa N-acetyltransferase PA4534.

The GCN5-related N-acetyltransferase (GNAT) superfamily includes a large and diverse group of enzymes that catalyzes the transfer of an acetyl group from acetyl coenzyme A (Ac-CoA) to the amine group of a substrate. Substrates include protein N-terminus, lysine of histone tails, and other small molecules such as aminoglycoside, serotonin, and glucose-6-phosphate. GNAT superfamily of proteins is involved in many physiologically important reactions in eukaryotes and prokaryotes. However, functions of many GNATs remain unknown and PA4534 is one of those uncharacterized GNAT proteins from Pseudomonas aeruginosa. To investigate functions of the PA4534, we determined the apo and Ac-CoA bound PA4534 structures. Our structures showed that PA4534 shared common characteristic structures with other GNAT family N-acetyltransferases and contained a potential substrate binding tunnel close to the bound Ac-CoA.

A novel esterase subfamily with α/ß-hydrolase fold suggested by structures of two bacterial enzymes homologous to L-homoserine O-acetyl transferases.

MekB from Pseudomonas veronii and CgHle from Corynebacteriumglutamicum belong to the superfamily of α/ß-hydrolase fold proteins. Based on sequence comparisons, they are annotated as homoserine transacetylases in popular databases like UNIPROT, PFAM or ESTHER. However, experimentally, MekB and CgHle were shown to be esterases that hydrolyse preferentially acetic acid esters. We describe the x-ray structures of these enzymes solved to high resolution. The overall structures confirm the close relatedness to experimentally validated homoserine acetyl transferases, but simultaneously the structures exclude the ability of MekB and CgHle to bind homoserine and acetyl-CoA. Insofar the MekB and CgHle structures suggest dividing the homoserine transacetylase family into subfamilies, namely genuine acetyl transferases and acetyl esterases with MekB and CgHle as constituting members of the latter.

Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation.

The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.

Identification of hydroxy fatty acid and triacylglycerol metabolism-related genes in lesquerella through seed transcriptome analysis.

BACKGROUND: Castor oil is the only commercial source of hydroxy fatty acid that has industrial value. The production of castor oil is hampered by the presence of the toxin ricin in its seed. Lesquerella seed also accumulates hydroxy fatty acid and is free of ricin, and thus it is being developed as a new crop for hydroxy fatty acid production. A high-throughput, large-scale sequencing of transcripts from developing lesquerella seeds was carried out by 454 pyrosequencing to generate a database for quality improvement of seed oil and other agronomic traits. Deep mining and characterization of acyl-lipid genes were conducted to uncover candidate genes for further studies of mechanisms underlying hydroxy fatty acid and seed oil synthesis. RESULTS: A total of 651 megabases of raw sequences from an mRNA sample of developing seeds was acquired. Bioinformatic analysis of these sequences revealed 59,914 transcripts representing 26,995 unique genes that include nearly all known seed expressed genes. Based on sequence similarity with known plant proteins, about 74% (19,861) genes matched with annotated coding genes. Among them, 95% (18,868) showed highest sequence homology with Arabidopsis genes, which will allow translation of genomics and genetics findings from Arabidopsis to lesquerella. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome assembly and identified 615 lesquerella genes involved in all known pathways of acyl-lipid metabolism. Further deep mining the transcriptome assembly led to identification of almost all lesquerella genes involved in fatty acid and triacylglycerol synthesis. Moreover, we characterized the spatial and temporal expression profiles of 15 key genes using the quantitative PCR assay. CONCLUSIONS: We have built a lesquerella seed transcriptome that provides a valuable reference in addition to the castor database for discovering genes involved in the synthesis of triacylglycerols enriched with hydroxy fatty acids. The information obtained from data mining and gene expression profiling will provide a resource not only for the study of hydroxy fatty acid metabolism, but also for the biotechnological production of hydroxy fatty acids in existing oilseed crops.

Toxicokinetics of novel psychoactive substances: characterization of N-acetyltransferase (NAT) isoenzymes involved in the phase II metabolism of 2C designer drugs.

The 2,5-dimethoxyphenethylamine-derived designer drugs (so-called "2Cs") recently became of great importance on the illicit drug market as stimulating hallucinogens. They are distributed and consumed as "novel psychoactive substances" (NPS) without any safety testing at the forefront. As previous studies have shown, the 2Cs are mainly metabolized by O-demethylation, N-acetylation, or deamination. Therefore, the aim of this study was to elucidate the role of the recombinant human N-acetyltransferase (NAT) isoforms 1 and 2 in the phase II metabolism of 2Cs. For these studies, cDNA-expressed recombinant human NATs were used and formation of metabolites after incubation was measured using GC-MS. NAT2 could be shown to be the only isoform catalyzing the reaction in vitro, hence it should be the only relevant enzyme for in vivo acetylation. In general, all metabolite formation reactions followed classic Michaelis-Menten kinetics and the affinity to human NAT2 was increasing with the volume of the 4-substituent. In consequence, a slow acetylator phenotype or inhibition of NAT2 could lead to decreased N-acetylation and might lead to an increased risk of side effects caused by these novel psychoactive substances.

S-palmitoylation regulates biogenesis of core glycosylated wild-type and F508del CFTR in a post-ER compartment.

Defects in CFTR (cystic fibrosis transmembrane conductance regulator) maturation are central to the pathogenesis of CF (cystic fibrosis). Palmitoylation serves as a key regulator of maturational processing in other integral membrane proteins, but has not been tested previously for functional effects on CFTR. In the present study, we used metabolic labelling to confirm that wild-type and F508del CFTR are palmitoylated, and show that blocking palmitoylation with the pharmacologic inhibitor 2-BP (2-bromopalmitate) decreases steady-state levels of both wild-type and low temperature-corrected F508del CFTR, disrupts post-ER (endoplasmic reticulum) maturation and reduces ion channel function at the cell surface. PATs (protein acyl transferases) comprise a family of 23 gene products that contain a DHHC motif and mediate palmitoylation. Recombinant expression of specific PATs led to increased levels of CFTR protein and enhanced palmitoylation as judged by Western blot and metabolic labelling. Specifically, we show that DHHC-7 (i) increases steady-state levels of wild-type and F508del CFTR band B, (ii) interacts preferentially with the band B glycoform, and (iii) augments radiolabelling by [3H]palmitic acid. Interestingly, immunofluorescence revealed that DHHC-7 also sequesters the F508del protein to a post-ER (Golgi) compartment. Our findings point to the importance of palmitoylation during wild-type and F508del CFTR trafficking.

Two fatty acid elongases possessing C18-Δ6/C18-Δ9/C20-Δ5 or C16-Δ9 elongase activity in Thraustochytrium sp. ATCC 26185.

Thraustochytrids, unicellular eukaryotic marine protists, accumulate polyunsaturated fatty acids. Here, we report the molecular cloning and functional characterization of two fatty acid elongase genes (designated tselo1 and tselo2), which could be involved in the desaturase/elongase (standard) pathway in Thraustochytrium sp. ATCC 26185. TsELO1, the product of tselo1 and classified into a Δ6 elongase group by phylogenetic analysis, showed strong C18-Δ6 elongase activity and relatively weak C18-Δ9 and C20-Δ5 activities when expressed in the budding yeast Saccharomyces cerevisiae. TsELO2, classified into a Δ9 elongase subgroup, showed only C16-Δ9 activity. When expressed in Aurantiochytrium limacinum mh0186 using a thraustochytrid-derived promoter and a terminator, TsELO1 exhibited almost the same specificity as expressed in the yeast but TsELO2 showed weak C18-Δ9 activity, in addition to its main C16-Δ9 activity. These results suggest that TsELO1 functions not only as a C18-Δ6 and a C20-Δ5 elongase in the main route but also as a C18-Δ9 elongase in the alternative route of standard pathway, while TsELO2 functions mainly as a C16-Δ9 elongase generating vaccenic acid (C18:1n-7) in thraustochytrids. This is the first report describing a fatty acid elongase harboring C16-Δ9 activity in thraustochytrids.

Proteome-wide analysis of amino acid variations that influence protein lysine acetylation.

Next-generation sequencing (NGS) technologies are yielding ever higher volumes of genetic variation data. Given this large amount of data, it has become both a possibility and a priority to determine what the functional implication of genetic variations is. Considering the essential roles of acetylation in protein functions, it is highly likely that acetylation related genetic variations change protein functions. In this work, we performed a proteome-wide analysis of amino acid variations that could potentially influence protein lysine acetylation characteristics in human variant proteins. Here, we defined the AcetylAAVs as acetylation related amino acid variations that affect acetylation sites or their interacting acetyltransferases, and categorized three types of AcetylAAVs. Using the developed prediction system, named KAcePred, we detected that 50.87% of amino acid variations are potential AcetylAAVs and 12.32% of disease mutations could result in AcetylAAVs. More interestingly, from the statistical analysis, we found that the amino acid variations that directly create new potential lysine acetylation sites have more chance to cause diseases. It can be anticipated that the analysis of AcetylAAVs might be useful to screen important polymorphisms and help to identify the mechanism of genetic diseases. A user-friendly web interface for analysis of AcetylAAVs is now freely available at http://bioinfo.ncu.edu.cn/AcetylAAVs_Home.aspx .

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system.

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins.

Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes.

BACKGROUND: Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. RESULTS: In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. CONCLUSIONS: There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should consider and, when possible take advantage of, the implications of polyploidy.

Molecular structure of WlbB, a bacterial N-acetyltransferase involved in the biosynthesis of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid .

The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 A resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the LbetaH superfamily of N-acyltransferases. Each subunit contains 27 beta-strands, 23 of which form the canonical left-handed beta-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O(delta1) of Asn 84 and the sugar C-3' amino group and the second between the backbone amide group of Arg 94 and the sugar C-5' carboxylate. The sugar C-3' amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.

Identification of novel acetyltransferase activity on the thermostable protein ST0452 from Sulfolobus tokodaii strain 7.

A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.