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

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  • Itoh Kunio
    Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University
  • Asakawa Tasuku
    Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University
  • Hoshino Kouichi
    Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University
  • Adachi Mayuko
    Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University
  • Fukiya Kensuke
    Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University
  • Watanabe Nobuaki
    Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi-Sankyo Co., Ltd.
  • Tanaka Yorihisa
    Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University

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Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie–Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater Vmax values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.

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