PDBsum entry 2jdd

Transferase

PDB id: 2jdd

Contents

Protein chain

145 a.a. *

Ligands

ACO

3PG-SO4

Waters ×129

* Residue conservation analysis

PDB id:

2jdd

Name:

Transferase

Title:

Glyphosate n-acetyltransferase bound to acetyl coa and 3- phosphoglycerate

Structure:

Glyphosate n-acetyltransferase. Chain: a. Engineered: yes

Source:

Bacillus licheniformis. Organism_taxid: 1402. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: the enzyme is a shuffled variant derived from genes discovered in b. Licheniformis

Resolution:

1.60Å R-factor: 0.157 R-free: 0.193

Authors:

D.L.Siehl,L.A.Castle,R.Gorton,R.J.Keenan

Key ref:

D.L.Siehl et al. (2007). The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase. J Biol Chem, 282, 11446-11455. PubMed id: 17272278 DOI: 10.1074/jbc.M610267200

Date:

06-Jan-07 Release date: 13-Feb-07

Protein chain

Q65LG7  (Q65LG7_BACLD) -  Probable acetyltransferase from Bacillus licheniformis (strain ATCC 14580 / DSM 13 / JCM 2505 / CCUG 7422 / NBRC 12200 / NCIMB 9375 / NCTC 10341 / NRRL NRS-1264 / Gibson 46)

Seq: 146 a.a.

Struc: 145 a.a.*

* PDB and UniProt seqs differ at 21 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1074/jbc.M610267200

J Biol Chem 282:11446-11455 (2007)

PubMed id: 17272278

The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase.

D.L.Siehl, L.A.Castle, R.Gorton, R.J.Keenan.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 11446-11455) copyright 2007.

Figures were selected by an automated process.