Regular Article
Structural Determination of the Substrate Specificities and Regioselectivities of the Rat and Human Fatty Acid ω-Hydroxylases☆

https://doi.org/10.1006/abbi.1999.1504Get rights and content

Abstract

The substrate and regiospecificities of the known CYP4A enzymes from rat (CYP4A1, -4A2, -4A3, and -4A8) and human (CYP4A11) have been determined using lauric (C12), myristic (C14), palmitic (C16), oleic (C18:1), and arachidonic (C20:4) acids. The CYP4A2 and CYP4A8 cDNAs required to complete the enzyme set were cloned from a rat kidney library. All five proteins were expressed in Escherichia coli and were purified with the help of a six-histidine tag at the carboxyl terminus. Two complementary CYP4A2-CYP4A3 chimeras fused at residue 119 (CYP4A2) and 122 (CYP4A3) were constructed to explore the roles of the 18 amino acid differences between the parent proteins in determining their catalytic profiles. The chimera in which the first 119 amino acids are from CYP4A2 indicates that the first 120 amino acids control the substrate specificity. The chimera in which the first 122 amino acids are from CYP4A3 is inactive due to a defect in electron transfer to the heme group. The highest activity for lauric acid was obtained with CYP4A1 and CYP4A8, but for all the proteins the activity decreased with increasing fatty acid chain length. The fact that none of the rat and human CYP4A enzymes exhibits a high activity with arachidonic acid appears to limit their role as catalysts for the physiologically important conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE).

References (48)

  • A.E.C.M. Simpson

    Gen. Pharmacol.

    (1997)
  • D. Kupfer

    Pharmacol. Ther.

    (1980)
  • C.N.A. Palmer et al.

    Biochim. Biophys. Acta

    (1993)
  • Y. Kikuta et al.

    FEBS Lett.

    (1994)
  • M.A. Alterman et al.

    Arch. Biochem. Biophys.

    (1995)
  • R.B. Bambal et al.

    Biochim. Biophys. Res. Commun.

    (1996)
  • P. Lu et al.

    Arch. Biochem. Biophys.

    (1997)
  • T. Aoyama et al.

    J. Lip. Res.

    (1990)
  • M.-H. Wang et al.

    Arch. Biochem. Biophys.

    (1996)
  • E.A. Dierks et al.

    J. Biol. Chem.

    (1998)
  • D.C. Muchmore et al.

    Methods Enzymol.

    (1989)
  • T. Omura et al.

    J. Biol. Chem.

    (1964)
  • P.R. Ortiz de Montellano et al.

    J. Biol. Chem.

    (1984)
  • R.T. Okita et al.

    Methods Enzymol.

    (1991)
  • F. Adas et al.

    J. Lipid Res.

    (1998)
  • C.N.A. Palmer et al.

    Biochim. Biophys. Acta

    (1993)
  • L.J. Roman et al.

    Arch. Biochem. Biophys.

    (1993)
  • G. Hosny et al.

    Arch. Biochem. Biophys.

    (1999)
  • J.C. McGiff et al.

    Steroids

    (1993)
  • P.W. Wertz et al.

    Biochim. Biophys. Acta

    (1987)
  • S. Kimura et al.

    DNA Cell Biol.

    (1989)
  • S. Kimura et al.

    DNA Cell Biol.

    (1989)
  • M. Stromstedt et al.

    DNA Cell Biol.

    (1990)

    • Cited by (0)

    ☆

    This work was supported by National Institutes of Health Grants GM25515 (PROM) and HL53994 (DLK).

    2

    To whom correspondence and reprint requests should be addressed at the Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94143-0446. Fax: (415) 502-4728. E-mail: [email protected].

    View full text
    Copyright © 2000 Academic Press. All rights reserved.