Human aldehyde oxidase (hAOX1): structure determination of the Moco‐free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms

Abstract

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 .

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

“…Moreover, the additives Gly-Gly-Gly ( T m of 59.72 °C), PEG3350 ( T m of 59.64 °C), DNA Library ( T m of 59.53 °C), Biotin ( T m of 59.31 °C), and TCEP ( T m of 59.29 °C) exhibit higher T m values than the control ( Figure 4 C). The observed slight positive melting temperature shift (Δ T m) might contribute to increase the protein stability reducing the respective conformational flexibility favoring further structural studies [ 57 , 58 ]. Hence, the referred additives could be considered promising candidates to be included in (i) the early stages of STEAP1 production and expression to promote a proper folding and to prevent aggregation, and (ii) the final protein buffer as putative crystallization additives.…”

“…Several other additives noticeably decreased the STEAP1 thermal stability, in particular Fos Choline 12 ( T m of 36.09 °C), K + Sulfate ( T m of 44.08 °C), Na + Phosphate (dibasic) ( T m of 46.60 °C), Mg 2+ Sulfate ( T m of 48.08 °C) and Na + Phosphate (monobasic) ( T m of 50.18 °C). These negative Δ T m values potentially indicate important structural changes towards a more disordered conformation or even protein misfolding [ 57 , 58 ]. A ranking of the best and the worst additives and their respective Tm is summed up in Figure 5 .…”

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case

“…The first three-dimensional (3D) structure of AOX was determined for the mouse isoform mAOX3 (PDB ID: 3ZYV), followed, in 2015, by the structure of hAOX1, reported in the free (PDB ID: 4UHW) and complex forms (structure with the inhibitor thioridazine and the substrate phthalazine bound, PDB ID: 4UHX). Later, structures of the single nucleotide polymorphs (SNPs) S1271L and G1269R were reported, and very recently the catalytic mechanism of hAOX1 for the oxidation reaction of phthalazine to phthalazin-1­(2 H )-one was studied with atomistic resolution using computational tools …”

Roles of selected non-P450 human oxidoreductase enzymes in protective and toxic effects of chemicals: review and compilation of reactions

“…Humans have functionally inactive AOX1 allelic variants as well as variants encoding enzymes with different catalytic activities (i.e., slow and rapid metabolizers) (Foti et al 2016 ; Hartmann et al 2016 ; Mota et al 2019 ). In addition, single nucleotide polymorphisms affecting the FAD binding site have been demonstrated to increase the rate of superoxide production (Foti et al 2017 ).…”