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

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.more » A crystal structure of BC showed the ATP analog AMPPCF{sub 2}P 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.« less

Authors:
; ; ; ; ; ;  [1]
  1. Pfizer
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1006907
Resource Type:
Journal Article
Journal Name:
Protein Sci.
Additional Journal Information:
Journal Volume: 17; Journal Issue: 2008
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ANTIBIOTICS; ATP; BACTERIA; BIOSYNTHESIS; BIOTIN; CARBOXYLASE; CARBOXYLATION; CARBOXYLIC ACIDS; CARRIERS; CATALYSIS; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; ENZYMES; HYPOTHESIS; KINETICS; MAGNESIUM IONS; MUTANTS; PROTEINS; REACTIVITY; SYNERGISM; TARGETS

Citation Formats

Mochalkin, Igor, Miller, J Richard, Evdokimov, Artem, Lightle, Sandra, Yan, Chunhong, Stover, Charles Ken, Waldrop, Grover L, and LSU). Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase. United States: N. p., 2008. Web. doi:10.1110/ps.035584.108.
Mochalkin, Igor, Miller, J Richard, Evdokimov, Artem, Lightle, Sandra, Yan, Chunhong, Stover, Charles Ken, Waldrop, Grover L, & LSU). Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase. United States. doi:10.1110/ps.035584.108.
Mochalkin, Igor, Miller, J Richard, Evdokimov, Artem, Lightle, Sandra, Yan, Chunhong, Stover, Charles Ken, Waldrop, Grover L, and LSU). Fri . "Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase". United States. doi:10.1110/ps.035584.108.
@article{osti_1006907,
title = {Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase},
author = {Mochalkin, Igor and Miller, J Richard and Evdokimov, Artem and Lightle, Sandra and Yan, Chunhong and Stover, Charles Ken and Waldrop, Grover L and LSU)},
abstractNote = {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 AMPPCF{sub 2}P 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.},
doi = {10.1110/ps.035584.108},
journal = {Protein Sci.},
number = 2008,
volume = 17,
place = {United States},
year = {2008},
month = {10}
}