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J. Biol. Chem., Vol. 280, Issue 47, 39363-39372, November 25, 2005

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Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis^*

From the School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia

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 Gly¹⁰⁸ or Gly²¹⁰ 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 Gly²¹⁰ 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.

Received for publication, August 22, 2005 , and in revised form, September 15, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 61-2-9385-2043; Fax: 61-2-9385-1483; E-mail: mythmok{at}mythmok.com.

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