Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Xanthine dehydrogenase (XDH) is a crucial enzyme in purine metabolism.
• Understanding XDH mutations aids in comprehending enzyme function and developing inhibitors.
• Aspergillus nidulans XDH shares homology with eukaryotic XDHs and bacterial aldehyde oxido-reductases (MOP).

Purpose of the Study:

• To characterize sequence changes in Aspergillus nidulans xanthine dehydrogenase (XDH) mutations.
• To map affected amino acids onto the 3D structure of related aldehyde oxido-reductase (MOP).
• To elucidate the functional consequences of these mutations, including cofactor interaction and inhibitor resistance.

Main Methods:

• Site-directed mutagenesis of Aspergillus nidulans xanthine dehydrogenase (XDH).
• Structural analysis by mapping mutations onto the 3D structure of Desulfovibrio gigas MOP.
• Biochemical assays to assess enzyme activity, cofactor binding, and inhibitor susceptibility.

Main Results:

• Two loss-of-function mutations were identified, affecting the molybdenum-pterin cofactor (MoCo) domain and substrate recognition domain.
• Mutations in two amino acids conferred resistance to the inhibitor allopurinol.
• Changes at Arg911 altered the hydroxylation site of 2-hydroxypurine from C-8 to C-6, a residue conserved in XDHs and MOP.
• Several mutations affected residues near the molybdenum center or its ligands.
• Arg911 and the conserved Glu833 were implicated in positioning purine substrates within the active site.

Conclusions:

• Amino acid substitutions in XDH significantly alter enzyme function, substrate specificity, and inhibitor interactions.
• Structural mapping provides insights into the roles of specific residues in cofactor binding and substrate orientation.
• These findings contribute to understanding the catalytic mechanism of XDH and related enzymes.