Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Authors

  • Igor Mochalkin,

    Corresponding author
    1. Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA
    • EMS, Pfizer Inc., Eastern Point Road, MS 8118W-352, Groton, CT 06340, USA; fax: (860) 686-1704.
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    • Present addresses: Pfizer, Inc., Groton, CT 06340, USA

  • J. Richard Miller,

    1. Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA
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    • Present addresses: Pfizer, Inc., Groton, CT 06340, USA

  • Artem Evdokimov,

    1. Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA
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    • Present addresses: Pfizer, Inc., Groton, CT 06340, USA

  • Sandra Lightle,

    1. Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA
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    • Pfizer, Inc., Chesterfield, MO 63017, USA

  • Chunhong Yan,

    1. Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA
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    • Bristol-Myers Squibb, PRI, Princeton, NJ 08543, USA

  • Charles Ken Stover,

    1. Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA
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    • Pfizer, Inc., Kalamazoo, MI 49007, USA

  • Grover L. Waldrop

    Corresponding author
    1. Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge, Louisiana 70803, USA
    • Department of Biological Sciences, Room 206 Life Sciences Building, Louisiana State University, Baton Rouge, LA 70803, USA; fax: (225) 578-7258.
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Abstract

Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or “flip-flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.

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