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<p>Cytochrome P450 comprises a group of monooxygenases that hydroxylate xenobiotics and natural compounds with diverse electron transfer systems. Here we identify a natural fusion protein of cytochrome (Cyt) <i>b</i><sub>5</sub> and Cyt <i>b</i><sub>5</sub> reductase (CBBR) that transfers electrons from NADH to the cytochrome P450 CYP540A2. This cytochrome P450 system hydroxylates medium-chain fatty acids (MCFAs) to generate (<i>R</i>)-β-hydroxy-MCFAs with 7–12 carbon atoms. Kinetic studies of CYP540A2 mutants indicate that side chains of Ser431 and Gln542 residues bind the carboxyl moiety of MCFAs for hydroxylation at their β-carbons. Pre-steady state kinetics also indicate that a predicted linker region between the FAD- and Cyt <i>b</i><sub>5</sub>-domains of CBBR modulates electron transfer from NADH to CYP540A2. The present study also identifies a dehydrogenase that oxidizes (<i>R</i>)-β-hydroxy-MCFAs to β-oxo-fatty acids that are substrates in the general β-oxidation mechanism of fatty acid degradation. The genes encoding CBBR, CYP540A2, and (<i>R</i>)-β-hydroxy-MCFA dehydrogenase are clustered in the genome of the fungus <i>Aspergillus nidulans</i> and related fungi. The <i>A. nidulans</i> genes are induced by MCFAs, and disrupting CBBR and CYP540A2 genes accumulated more intracellular decanoic acid. Our findings reveal an adaptive monooxygenase-dependent β-oxidation mechanism that alternates with conventional β-oxidation, thus allowing fungi to metabolize MCFAs.</p>

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Unique electron transfer system of cytochrome P450 monooxygenase includes a mechanism of fatty acid β-oxidation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月25日

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  • Saito Kojima,
  • Kyosei Shinji,
  • Hana Namiki,
  • Kouta Suzuki,
  • Motoyuki Shimizu,
  • Chihiro Kadooka,
  • Nozomi Katsuki,
  • Shunsuke Masuo,
  • Madoka Amahisa,
  • Yuki Doi,
  • Norio Takeshita,
  • Naoki Takaya

摘要

Cytochrome P450 comprises a group of monooxygenases that hydroxylate xenobiotics and natural compounds with diverse electron transfer systems. Here we identify a natural fusion protein of cytochrome (Cyt) b5 and Cyt b5 reductase (CBBR) that transfers electrons from NADH to the cytochrome P450 CYP540A2. This cytochrome P450 system hydroxylates medium-chain fatty acids (MCFAs) to generate (R)-β-hydroxy-MCFAs with 7–12 carbon atoms. Kinetic studies of CYP540A2 mutants indicate that side chains of Ser431 and Gln542 residues bind the carboxyl moiety of MCFAs for hydroxylation at their β-carbons. Pre-steady state kinetics also indicate that a predicted linker region between the FAD- and Cyt b5-domains of CBBR modulates electron transfer from NADH to CYP540A2. The present study also identifies a dehydrogenase that oxidizes (R)-β-hydroxy-MCFAs to β-oxo-fatty acids that are substrates in the general β-oxidation mechanism of fatty acid degradation. The genes encoding CBBR, CYP540A2, and (R)-β-hydroxy-MCFA dehydrogenase are clustered in the genome of the fungus Aspergillus nidulans and related fungi. The A. nidulans genes are induced by MCFAs, and disrupting CBBR and CYP540A2 genes accumulated more intracellular decanoic acid. Our findings reveal an adaptive monooxygenase-dependent β-oxidation mechanism that alternates with conventional β-oxidation, thus allowing fungi to metabolize MCFAs.