Kinetic and physical characterization of the inducible UDP-N-acetylglucosamine pyrophosphorylase from Giardia intestinalis.
The UDP-N-acetylglucosamine pyrophosphorylase in Giardia intestinalis (GiUAP) is one of the five inducible enzymes to synthesize UDP-GalNAc, which is an important precursor for cyst wall synthesis. The recombinant UDP-N-acetylglucosamine pyrophosphorylase (rGiUAP) and its mutants G108A and G210A were expressed and identified by SDS-PAGE, size-exclusion chromatography, Western hybridization, and MALDI mass spectrometry. Sequence comparison with other eukaryotic UAPs has identified three specific motifs. Within these motifs alanine substitution for Gly(108) or Gly(210) dramatically reduced the pyrophosphate synthesis, suggesting these amino acids are catalytic residues. Besides, the rGiUAP was found to have relaxed binding to other uridine-based nucleotides, suggesting the substrate binding pocket is specific to uridine rather than phosphate group(s). Moreover, thermal denaturation analysis showed a significant increase in T(m) for the rGiUAP and G108A upon binding of the substrate Mg-UTP. In contrast, G210A showed a decreased T(m) upon binding of Mg-UTP. These results showed that binding of Mg-UTP increases protein stability of the rGiUAP, and the catalytic residue Gly(210) plays a significant role in stabilizing the protein structure. Such stabilization effect induced by substrate binding might be physiologically important as it favors the production of UDP-GlcNAc and hence the downstream GalNAc, which is crucial to survival of Giardia. These results help to define the essential amino acids for catalysis in the GiUAP and reveal the role of Mg-UTP binding in regulation of protein stability.
Mok
Myth T S
MT
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia. mythmok@mythmok.com
Edwards
Michael R
MR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004790:Enzyme Induction; D004795:Enzyme Stability; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014544:Uridine Triphosphate / Q000378:metabolism
D016054:DNA, Protozoan; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D014544:Uridine Triphosphate