TY  - JOUR
A1  - Foti, Alessandro
A1  - Hartmann, Tobias
A1  - Coelho, Catarina
A1  - Santos-Silva, Teresa
A1  - Romao, Maria Joao
A1  - Leimkühler, Silke
T1  - Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms
JF  - Drug metabolism and disposition : the biological fate of chemicals
N2  - Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme’s role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.
Y1  - 2016
U6  - https://doi.org/10.1124/dmd.115.068395
SN  - 0090-9556
SN  - 1521-009X
VL  - 44
SP  - 1277
EP  - 1285
PB  - American Society for Pharmacology and Experimental Therapeutics
CY  - Bethesda
ER  - 
TY  - JOUR
A1  - Coelho, Catarina
A1  - Foti, Alessandro
A1  - Hartmann, Tobias
A1  - Santos-Silva, Teresa
A1  - Leimkühler, Silke
A1  - Romao, Maria Joao
T1  - Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase
JF  - Nature chemical biology
N2  - Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.
Y1  - 2015
U6  - https://doi.org/10.1038/NCHEMBIO.1895
SN  - 1552-4450
SN  - 1552-4469
VL  - 11
IS  - 10
SP  - 779
EP  - +
PB  - Nature Publ. Group
CY  - New York
ER  -