Characterization of human aldehyde oxidase: substrate specificities, mode of inhibition and superoxide production

• Applicant: Professorin Dr. Silke Leimkühler
• Subject Area: Biochemistry
• Term: from 2012 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 224728554

Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. So far, the physiological function of AOX for mammals is unknown. However, human AOX1 is of increasing pharmacological interest due to its recognized role in phase I drug metabolism. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. We established a codon-optimized heterologous expression system for human AOX1 in Escherichia coli. This expression system now enables us to obtain sufficient amounts of active protein for kinetic characterizations and sets the basis for site-directed mutagenesis and structure-function studies. To define the physiological function of human AOX1 the active site will be investigated in detail. We plan to characterize selected single polynucleotide polymorphisms (SNPs) identified around the substrate and inhibitor binding site in human individuals and analyze their effect on hAOX1 activity. We will further introduce amino acid exchanges around the FAD site and will analyze the effect on intramolecular electron transfer. Further, we will analyze the role of hAOX1 in the production of reactive oxygen species (ROS), since hAOX1 as a true oxidase was suggested to significantly contribute to the production of high amounts of endogenous O2-. The co-crystal structures of hAOX1 with substrates and inhibitors will be solved and will help to understand the mechanism of substrate and inhibitor binding for future drug developments. Our studies will be particularly important to define the physiopathological functions of AOX1 and will help to understand the drug metabolizing role of AOX1 for humans in general.

• DFG Programme: Research Grants