Functional Analysis of Aldehyde Oxidase Using Expressed Chimeric Enzyme between Monkey and Rat

Abstract

Aldehyde oxidase (AO, EC 1.2.3.1) and xanthine oxidase are major members of the molybdenum hydroxylase family. Both enzymes consist of a homodimer with a subunit molecular mass of about 150 kDa. Each subunit is made up of a 20 kDa domain containing two different 2Fe-2S clusters in which reducing equivalents necessary for catalysis are stored, a 40 kDa domain containing a flavine adenine dinucleotide (FAD), and an 80 kDa domain containing a molybdenum cofactor (MoCo) in which a substrate binding site is located. 1)AO catalyzes the oxidation of a wide range of endogenous and exogenous aldehydes and N-heterocyclic aromatic compounds. The representative N-heterocyclic-containing drugs that serve as substrates for AO are famciclovir, methotrexate, 6-mercaptopurine, and cinchona alkaloids. [1][2][3][4][5][6] In addition, the atypical antipsychotic drug, ziprasidone, is mainly metabolized by AO-catalyzed reductive ring cleavage in human. 7,8) AO has several pharmacokinetically interesting properties of remarkable species differences, large strain differences in rat, and individual differences in some kinds of rat strains. Among those phenomena, species differences have been widely demonstrated in the metabolism of many drugs catalyzed by AO. They include an ocular hypotensive agent, brimonidine, 9) antiviral drugs, famciclovir and 6-deoxypenciclovir, 10) an antineoplastic drug, methotrexate, 11) an ultrashort acting hypnotic, zaleplon, 12) and an oral antitumor agent, zebularine.13) Thus, it is now well established that the variations in AO activity may be dependent on not only animal species, but also on the chemical structure of the substrate. Roughly speaking, AO activity is high in monkeys and humans, moderate to low in rats and mice, and deficient in dogs. Similar to those reports, we observed remarkable species differences, rat strain differences, and polymorphism-based individual differences in Donryu strain rat in the AO-catalyzed 2-oxidation activity of the (S)-enantiomer of RS-8359,14-18) The compound is a reversible and selective monoamine oxidase (MAO)-A inhibitor 19,20) and has been developed as an anti-depressant. 21,22) As to species differences, monkeys showed the highest activity followed by humans. Furthermore, monkey and human liver cytosol preparations exhibited a biphasic pattern in the Eadie-Hofstee plots for the 2-oxidation activity of (S)-RS-8359, but not rat and mouse liver cytosols. The expression system of monkey AO full cDNA suggested that the biphasic Eadie-Hofstee profile might be produced by the existence of two active sites in a single AO enzyme rather than by the existence of two AO isoforms. 23)Molecular cloning of AO cDNA has been accomplished in mouse, 24,25) rat, 26) rabbit, 27) bovine, 28) and human. 29-31) The deduced primary structure of AO proteins have been characterized with regard to consensus sequences for two distinct 2Fe-2S clusters and five MoCo-binding sites. Wright et al. 26) indicated that the kinetic differences of AO activity between male and female rats are due...

Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity

“…This is in accordance with Moco-domain swop experiments performed with monkey and rat AOX orthologs [29]. A correlation of k cat and the difference in reduction potential between the Mo V/VI pair and FeSI was observed.…”

“…A linear correlation according to the Marcus theory was only observed for the reduction potential difference between the MoV/VI redox pair and FeSI, with values for the electronic coupling matrix of H AB  = 5.76×10 −16 ±2.14×10 −16 and reorganization energy of λ  = 2.57×10 4 ±6.84×10 3 J/mol. A previous report by Itoh et al [29] examined the effect of Moco domain swop between monkey and rat AOX on the conversion of (S)-RS-8359. The authors observed that the substrate affinity and susceptibility to substrate inhibition of the chimeric variants was equal to the enzyme from which the Moco domain originated.…”

Steady-State Kinetic Mechanism and Reductive Half-Reaction of d-Arginine Dehydrogenase from Pseudomonas aeruginosa

“…Both these possibilities appear very unlikely since the available crystallographic structure of the enzyme cocrystallized with d -arginine shows that the active site cavity of the enzyme is almost entirely occupied with a single iminoarginine, thereby preventing the binding of a second arginine . Inhibition of catalytic turnover due to formation of a dead-end complex between the substrate and the reduced form of the enzyme was previously observed in other flavoproteins, such as nitroalkane oxidase , aldehyde oxidase , flavocytochrome b 2 , cellobiose dehydrogenase , and thymidylate synthase .…”