Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - The N-acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of glycosylphosphatidylinositol biosynthesis is a zinc metalloenzyme
AU - Urbaniak, Michael D.
AU - Crossman, Arthur
AU - Chang, Tunhan
AU - Smith, Terry K.
AU - van Aalten, Daan M. F.
AU - Ferguson, Michael A. J.
PY - 2005/6/17
Y1 - 2005/6/17
N2 - 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.
AB - 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.
KW - Acetylglucosamine
KW - Alanine
KW - Amidohydrolases
KW - Amino Acid Sequence
KW - Animals
KW - Antigens, CD55
KW - Binding Sites
KW - Cations
KW - Cell-Free System
KW - Chromatography, High Pressure Liquid
KW - Crystallography, X-Ray
KW - Electrophoresis, Polyacrylamide Gel
KW - Escherichia coli
KW - Flow Cytometry
KW - Genetic Complementation Test
KW - Glycosylphosphatidylinositols
KW - Green Fluorescent Proteins
KW - Ions
KW - Models, Molecular
KW - Molecular Sequence Data
KW - Mutagenesis, Site-Directed
KW - Mutation
KW - Phosphatidylinositols
KW - Plasmids
KW - Point Mutation
KW - Protein Conformation
KW - Protein Structure, Tertiary
KW - Rats
KW - Recombinant Fusion Proteins
KW - Recombinant Proteins
KW - Sequence Homology, Amino Acid
KW - Transfection
KW - Trypanosoma brucei brucei
KW - Zinc
U2 - 10.1074/jbc.M502402200
DO - 10.1074/jbc.M502402200
M3 - Journal article
C2 - 15817455
VL - 280
SP - 22831
EP - 22838
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 24
ER -