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Structure and substrate‐based inhibitor of Aspergillus fumigatus UDP‐ N ‐acetylglucosamine pyrophosphorylase ( Af UAP1)
Author(s) -
Raimi Olawale Ganiyu,
HurtadoGuerrero Ramon,
Ibrahim Adel,
Borodkin Vladimir,
Urbaniak Micheal,
Mariño Karina,
Mariño Karina,
Ferguson Micheal,
vanAalten Daan
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.714.6
UDP‐ N ‐acetylglucosamine pyrophosphorylase (UAP1) is the last of the four enzymes responsible for the biosynthesis of uridine diphosphate‐ N ‐acetylglucosamine (UDP‐GlcNAc) in eukaryotes. The enzyme in the presence of UTP reversibly converts GlcNAc‐1P to sugar nucleotide (UDP‐GlcNAc). This experiment was aimed at obtaining structural information, elucidating the reaction mechanism and identifying a potent inhibitor for Af UAP1. Af UAP1 was cloned as a GST‐Fusion protein and expressed in E. coli . Crystallization of the protein was achievable after the removal of 27 amino acid residues from the N‐terminus. A structural snapshot of UDP‐GlcNAc, pyrophosphate and Mg 2+ complex provides the first Michaelis complex trapped for this enzyme, revealing the structural basis of the previously reported Mg 2+ dependency. This structure reveals pentacoordinated magnesium ion and explains why this metal is essential for catalysis. UTP‐analogue (α, β‐methylenephosphonate) was found to inhibit Af UAP1 with an IC 50 of 115 ± 40 μM. The detailed mechanistic insight and inhibitor provided by this work will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential drug target. This work was supported by Welcome trust and European Synchrotron radiation facility (ESRF), Grenoble, France.