Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

Abstract

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30-40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

Deficiency of AtGFAT1 activity impairs growth, pollen germination and tolerance to tunicamycin in Arabidopsis

“…T-DNA insertions in the exon of AtGFAT1 had a lethal effect (Table 1; Fig. 2A), which is consistent with previous reports showing the lethal phenotype of Arabidopsis mutants lacking glucosamine-6-phosphate N -acetyltransferase (GNA) and N -acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT) activity catalysing the second and last step of HBP, respectively (Riegler et al , 2012; Chen et al , 2014). However, T-DNA insertion in OsGNA1 of rice caused a defect in root elongation, but not a lethal phenotype (Jiang et al , 2005).…”

“…Recent work on genes encoding enzymes for the second and last steps of the HBP in plants have shown that the HBP is indispensable for plant growth and development, as in animals. For example, single base and knockout mutations in the GNA gene in Arabidopsis, encoding GlcN-6-P N -acetyltransferase, resulted in growth retardation and a lethal phenotype, respectively (Nozaki et al ., 2012; Riegler et al , 2012). The T-DNA insertion in GNA in rice reduced the wild-type UDP-GlcNAc content by about 90%, giving rise to abnormal root morphology (Jiang et al , 2005).…”

Identification of anN‐acetylglucosamine kinase essential for UDP‐N‐acetylglucosamine salvage synthesis inArabidopsis

“…The pathway of de novo UDP‐GlcNAc synthesis originates from Fru‐6P, a glycolytic intermediate. l ‐Glutamine d ‐fructose‐6‐phosphate amidotransferase (GFAT), GNA1, and N ‐acetylglucosamine‐1‐phosphate uridylyltransferase (GlcNAc1pUT) are involved in this pathway[18–20]. GNK phosphorylates GlcNAc and produces GlcNAc‐6P, an intermediate of the de novo pathway.…”

Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

“…However, the acceptor substrate in the reaction catalyzed by ScGNA1 (GlcN6P) has a much lower p K a (7.75) than that of other GNAT acceptor substrates and is likely to bind to the enzyme in a deprotonated form, eliminating the need for a general base [ 99 ]. The structures of the ScGNA1 homologs from Homo sapiens [ 100 , 101 ], Aspergillus fumigatus [ 100 ], Arabidopsis thaliana [ 102 ] and C. elegans [ 103 ] were also reported. The main-chain oxygen of E156 of human GNA1 (hGNA1) forms a hydrogen bond with the GlcN6P’s amino group and thus increases its nucleophilic nature.…”