TY  - JOUR
A1  - Paragas, Erickson M.
A1  - Humphreys, Sara C.
A1  - Min, Joshua
A1  - Joswig-Jones, Carolyn A.
A1  - Leimkühler, Silke
A1  - Jones, Jeffrey P.
T1  - ecoAO
BT  - a simple system for the study of human aldehyde oxidases role in drug metabolism
JF  - ACS OMEGA
N2  - Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO’s preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.
Y1  - 2017
U6  - https://doi.org/10.1021/acsomega.7b01054
SN  - 2470-1343
VL  - 2
SP  - 4820
EP  - 4827
PB  - American Chemical Society
CY  - Washington
ER  - 
TY  - CHAP
A1  - Leimkühler, Silke
A1  - Hartmann, Tobias
A1  - Garattini, Enrico
A1  - Jones, Jeffrey P.
T1  - Structure-function studies on human aldehyde oxidase and the impact of polymorphisms on enzyme activity
T2  - Drug metabolism reviews : biotransformation and disposition of xenobiotics ; official journal of the International Society for the Study of Xenobiotics
Y1  - 2011
SN  - 0360-2532
VL  - 43
IS  - 6
SP  - 13
EP  - 13
PB  - Taylor & Francis Group
CY  - London
ER  - 
TY  - JOUR
A1  - Hartmann, Tobias
A1  - Terao, Mineko
A1  - Garattini, Enrico
A1  - Teutloff, Christian
A1  - Alfaro, Joshua F.
A1  - Jones, Jeffrey P.
A1  - Leimkühler, Silke
T1  - The impact of single nucleotide polymorphisms on human aldehyde oxidase
JF  - Drug metabolism and disposition : the biological fate of chemicals
N2  - 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, N-1-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 mu g/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.
Y1  - 2012
U6  - https://doi.org/10.1124/dmd.111.043828
SN  - 0090-9556
VL  - 40
IS  - 5
SP  - 856
EP  - 864
PB  - American Society for Pharmacology and Experimental Therapeutics
CY  - Bethesda
ER  -