title: The N-acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of glycosylphosphatidylinositol biosynthesis is a zinc metalloenzyme.
abstract: The de-N-acetylation of N-acetyl-D-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn(2+) > Cu(2+) > Ni(2+) > Co(2+) > Mg(2+). Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.
authors.affiliation: Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, The University of Dundee, Scotland, UK.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Urbaniak
authors.forename: Arthur
authors.initials: A
authors.lastname: Crossman
authors.forename: Tunhan
authors.initials: T
authors.lastname: Chang
authors.forename: Terry K
authors.initials: TK
authors.lastname: Smith
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000581:Amidohydrolases / Q000737:chemistry* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018958:CD55 Antigens / Q000096:biosynthesis; D002412:Cations; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005816:Genetic Complementation Test; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007477:Ions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010716:Phosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry*
chemical_list: D018958:CD55 Antigens; D002412:Cations; D017261:Glycosylphosphatidylinositols; D007477:Ions; C081027:N-acetylglucosaminylphosphatidylinositol; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000581:Amidohydrolases; C087696:N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase; D015032:Zinc; D000409:Alanine; D000117:Acetylglucosamine