title: 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.
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany. riegler@mpimp-golm.mpg.de
authors.forename: Heike
authors.initials: H
authors.lastname: Riegler
authors.forename: Thomas
authors.initials: T
authors.lastname: Herter
authors.forename: Irina
authors.initials: I
authors.lastname: Grishkovskaya
authors.forename: Anja
authors.initials: A
authors.lastname: Lude
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Ryngajllo
authors.forename: Marie E
authors.initials: ME
authors.lastname: Bolger
authors.forename: Bernd
authors.initials: B
authors.lastname: Essigmann
authors.forename: Björn
authors.initials: B
authors.lastname: Usadel
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D018360:Crystallography, X-Ray; D018506:Gene Expression Regulation, Plant; D051081:Glucosamine 6-Phosphate N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D051081:Glucosamine 6-Phosphate N-Acetyltransferase