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PDBsum entry 2jdd
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References listed in PDB file
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Key reference
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Title
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The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase.
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Authors
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D.L.Siehl,
L.A.Castle,
R.Gorton,
R.J.Keenan.
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Ref.
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J Biol Chem, 2007,
282,
11446-11455.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
perfect match.
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Abstract
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GAT is an N-acetyltransferase from Bacillus licheniformis that was optimized by
gene shuffling for acetylation of the broad spectrum herbicide, glyphosate,
forming the basis of a novel mechanism of glyphosate tolerance in transgenic
plants (Castle, L. A., Siehl, D. L., Gorton, R., Patten, P. A., Chen, Y. H.,
Bertain, S., Cho, H. J., Duck, N., Wong, J., Liu, D., and Lassner, M. W. (2004)
Science 304, 1151-1154). The 1.6-A resolution crystal structure of an optimized
GAT variant in ternary complex with acetyl coenzyme A and a competitive
inhibitor, 3-phosphoglyerate, defines GAT as a member of the GCN5-related family
of N-acetyltransferases. Four active site residues (Arg-21, Arg-73, Arg-111, and
His-138) contribute to a positively charged substrate-binding site that is
conserved throughout the GAT subfamily. Structural and kinetic data suggest that
His-138 functions as a catalytic base via substrate-assisted deprotonation of
the glyphosate secondary amine, whereas another active site residue, Tyr-118,
functions as a general acid. Although the physiological substrate is unknown,
native GAT acetylates D-2-amino-3-phosphonopropionic acid with a kcat/Km of 1500
min-1 mM-1. Kinetic data show preferential binding of short analogs to native
GAT and progressively better binding of longer analogs to optimized variants.
Despite a 200-fold increase in kcat and a 5.4-fold decrease in Km for
glyphosate, only 4 of the 21 substitutions present in R7 GAT lie in the active
site. Single-site revertants constructed at these positions suggest that
glyphosate binding is optimized through substitutions that increase the size of
the substrate-binding site. The large improvement in kcat is likely because of
the cooperative effects of additional substitutions located distal to the active
site.
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Figure 2.
FIGURE 2. A, structure of R7 GAT bound to acetyl coenzyme A
(ACO, magenta) and the competitive inhibitor, 3PG (green). B,
molecular surface of the R7 GAT ternary complex colored by its
electrostatic potential reveals the electropositive
substrate-binding site. Acetyl coenzyme A and 3PG are shown in
the active site cleft. C, close up of the active site. Polar
contacts made by 3PG are indicated (gray dashes), and water
molecules are shown as red spheres. Modeling glyphosate into the
active site positions its 2° amine (equivalent position on
3PG marked with an asterisk) within 3.8 Å of the carbonyl
carbon of AcCoA. D, shuffling changes are distributed throughout
the enzyme. A C-  trace indicates the
locations of 21 amino acid substitutions (yellow and blue
spheres) made between native and R7 GAT. Substitutions located
within 5 Å of either ligand are shown in blue.
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Figure 3.
FIGURE 3. A, plots of k[cat] versus pH for N-acetylation of
glyphosate by R7 (  ) and R7-Y118F (  ) GAT.
The pK[1] and pK[2] values shown on the graphs refer to the
acidic and basic limbs of the profiles, respectively. See
"Experimental Procedures" for experimental conditions and
theoretical fits. B, proposed chemical mechanism for
N-acetylation of glyphosate by GAT.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
11446-11455)
copyright 2007.
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