 |
PDBsum entry 3g8c
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Crystal structure of biotin carboxylase in complex with substrates and implications for its catalytic mechanism.
|
|
|
Authors
|
|
C.Y.Chou,
L.P.Yu,
L.Tong.
|
|
|
Ref.
|
|
J Biol Chem, 2009,
284,
11690-11697.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Biotin-dependent carboxylases are widely distributed in nature and have
important functions in many cellular processes. These enzymes share a conserved
biotin carboxylase (BC) component, which catalyzes the ATP-dependent
carboxylation of biotin using bicarbonate as the donor. Despite the availability
of a large amount of biochemical and structural information on BC, the molecular
basis for its catalysis is currently still poorly understood. We report here the
crystal structure at 2.0 A resolution of wild-type Escherichia coli BC in
complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure
suggests that Glu(296) is the general base that extracts the proton from
bicarbonate, and Arg(338) is the residue that stabilizes the enolate biotin
intermediate in the carboxylation reaction. The B domain of BC is positioned
closer to the active site, leading to a 2-A shift in the bound position of the
adenine nucleotide and bringing it near the bicarbonate for catalysis. One of
the oxygen atoms of bicarbonate is located in the correct position to initiate
the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This
oxygen is also located close to the N1' atom of biotin, providing strong
evidence that the phosphate group, derived from decomposition of
carboxyphosphate, is the general base that extracts the proton on this N1' atom.
The structural observations are supported by mutagenesis and kinetic studies.
Overall, this first structure of BC in complex with substrates offers
unprecedented insights into the molecular mechanism for the catalysis by this
family of enzymes.
|
|
|
|
|
|
|
|
Figure 1.
Structure of wild-type E. coli BC in complex with biotin,
bicarbonate, and Mg-ADP. A, overall structure of the E. coli BC
dimer in complex with the substrates. Biotin, bicarbonate, and
ADP are shown in pink, black, and green for carbon atoms,
respectively. The modeled binding mode of ATP (in gray) is shown
for the monomer in yellow (PDB 3G8C). B, final 2F[o] - F[c]
electron density for biotin at 2.0 Å resolution, contoured
at 1σ. The omit F[o] - F[c] density looks essentially the same.
C, electron density for bicarbonate. D, electron density for
Mg-ADP. E, schematic drawing showing detailed interactions in
the active site of BC. Hydrogen bonding and ion pair
interactions are indicated with the dashed lines in red. All
structure figures in this paper are produced with PyMOL (44).
|
|
Figure 3.
Molecular mechanism for the catalysis by BC. A, modeled
binding mode of ATP (in gray) in the active site of BC. The
bicarbonate is in the appropriate location to initiate the
reaction. B, schematic drawing of the catalytic mechanism of BC.
Glu^296 and Arg^338 have crucial roles in this reaction.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
11690-11697)
copyright 2009.
|
|
|
|
|
|
|
