The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase

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-angstrom 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 k(cat)/K-m 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 k(cat) and a 5.4-fold decrease in K-m 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.