Lipids

, Volume 34, Issue 8, pp 841–846

Characterization of the ybdT gene product of Bacillus subtilis: Novel fatty acid β-hydroxylating cytochrome P450

  • Isamu Matsunaga
  • Atsuo Ueda
  • Nagatoshi Fujiwara
  • Tatsuo Sumimoto
  • Kosuke Ichihara
Article

Abstract

We have characterized the gene encoding fatty acid α-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce β-hydroxyristic acid and α-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of β-hydroxyl product was slightly higher than that of α-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the β-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of β-hydroxy fatty acid.

Abbreviations

ADAM

9-anthryldiazomethane

GC-MS

gas chromatography-mass spectrometry

HPLC

high-performance liquid chromatography

P450

cytochrome P450

P450BSβ

fatty acid β-hydroxylating P450 from Bacillus subtilis

P450SPα

fatty acid α-hydroxylating P450 from Sphingomonas paucimobilis

TMS

trimethylsilyl

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Copyright information

© AOCS Press 1999

Authors and Affiliations

  • Isamu Matsunaga
    • 3
  • Atsuo Ueda
    • 3
  • Nagatoshi Fujiwara
    • 1
  • Tatsuo Sumimoto
    • 3
    • 2
  • Kosuke Ichihara
    • 3
  1. 1.Department of BacteriologyOsaka City University Medical SchoolOsakaJapan
  2. 2.Osaka Prefectural Institute of Public HealthOsakaJapan
  3. 3.Department of Molecular RegulationOsaka City University Medical SchoolOsakaJapan

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