 |
PDBsum entry 1n0h
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
The crystal structures of klebsiella pneumoniae acetolactate synthase with enzyme-Bound cofactor and with an unusual intermediate.
|
|
|
Authors
|
|
S.S.Pang,
R.G.Duggleby,
R.L.Schowen,
L.W.Guddat.
|
|
|
Ref.
|
|
J Biol Chem, 2004,
279,
2242-2253.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine
diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of
pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a
second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants,
fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino
acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a
catabolic enzyme required for the butanediol fermentation, and does not contain
FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS.
The overall structure is similar to AHAS except for a groove that accommodates
FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP
cofactor has an unusual conformation that is unprecedented among the 26 known
three-dimensional structures of nine ThDP-dependent enzymes, including AHAS.
This conformation suggests a novel mechanism for ALS. A second structure, at 2.0
A, is described in which the enzyme is trapped halfway through the catalytic
cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The
cofactor has a tricyclic structure that has not been observed previously in any
ThDP-dependent enzyme, although similar structures are well known for free
thiamine. This structure is consistent with our proposed mechanism and probably
results from an intramolecular proton transfer within a tricyclic carbanion that
is the true reaction intermediate. Modeling of the second molecule of pyruvate
into the active site of the enzyme with the bound intermediate is consistent
with the stereochemistry and specificity of ALS.
|
|
|
|
|
|
|
|
Figure 2.
FIG. 2. Structure of K. pneumoniae ALS. A shows a ribbon
diagram of the overall structure of the resting enzyme tetramer,
with the monomers colored green (monomer A), red (B), blue (C),
and yellow (D). There is a vertical 2-fold axis of symmetry in
this view. The asymmetric unit contains monomers A and B,
whereas the active sites are at the AC and BD interfaces.
Monomer A is shown in B, with cylinder representations of  -helices
(red) and  -strands (turquoise)
shown as arrows, connected by random coil (green). C and D
compare the -domains of ALS (C) and
AHAS (D), with secondary structure indicated as in B. The
residues in contact with FAD (stick model, D) in AHAS and their
structural equivalents in ALS (C) are shown in surface
representation.
|
|
Figure 7.
FIG. 7. The active sites of ALS and AHAS. A shows the
active site region of K. pneumoniae ALS (resting enzyme), and B
shows the active site of yeast AHAS viewed in a similar
orientation. Residues with and without the prime symbol are
derived from different monomers. C, the active site of ALS is
shown with the second molecule of pyruvate modeled in so that it
makes favorable contacts and is orientated so that it would
yield the S-enantiomer of acetolactate (D). In this model, the
intermediate is represented as the tricyclic carbanion (IVb,
Fig. 5), and an alternate conformation of Lys36 is shown for
which the  -amino group forms an
ionic interaction with the carboxylate of the second pyruvate.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
2242-2253)
copyright 2004.
|
|
|
|
|
|
|
