Human aldehyde oxidase ( hAOX 1): structure determination of the Moco‐free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms

Human aldehyde oxidase ( hAOX 1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX 1 metabolizes different classes of xenobiotics and is one of the main drug‐metabolizing enzymes in the liver, along with cytochrome P450. hAOX 1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug‐metabolizing enzymes caused by single nucleotide polymorphisms ( SNP s) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)‐free form of hAOX 1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild‐type hAOX 1 and hAOX 1 with various SNP s (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and Thermo FAD , revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 °C. These results correlated with biochemical and structural data and enhance our understanding of hAOX 1 and the effect of SNP s in the gene encoding this enzyme in the human population. Enzymes Aldehyde oxidase ( EC1.2.3.1 ); xanthine dehydrogenase ( EC1.17.1.4 ); xanthine oxidase ( EC1.1.3.2 ). Databases Structural data are available in the Protein Data Bank under the accession number 6Q6Q .