Abstract
Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 μg/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.
Footnotes
This work was supported by the Cluster of Excellence “Unifying Concepts in Catalysis” (to C.T. and S.L.) coordinated by the Technische Universität Berlin and funded by the Deutsche Forschungsgemeinschaft and by grants from the Associazione Italiana per la Ricerca contro il Cancro and the Fondazione Italo Monzino (to E.G.).
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ABBREVIATIONS:
- AO
- aldehyde oxidase
- AOX1
- aldehyde oxidase 1
- FM
- fast metabolizer
- MCSF
- Moco sulfurase
- Moco
- molybdenum cofactor
- PAGE
- polyacrylamide gel electrophoresis
- PCR
- polymerase chain reaction
- PM
- poor metabolizer
- SNP
- single nucleotide polymorphism
- XDH
- xanthine dehydrogenase
- XO
- xanthine oxidase
- Vis
- visible
- MPT
- molybdopterin.
- Received November 10, 2011.
- Accepted January 25, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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