The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

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 ( Glyphosate (N-phosphonomethylglycine) is a widely used herbicide that acts nonselectively through inhibition of 5-enolpyruvylshikimate-3-phosphate (EPSP) 4 synthase in the aromatic biosynthesis pathway (2). Its efficacy against all plant species, low cost, low mammalian toxicity, and benign environmental impact favor its use in crops that have a transgenic tolerance mechanism (3). The predominant transgene currently in use encodes a bacterial EPSP synthase that is nearly insensitive to inhibition by glyphosate (2). We reported an alternative strategy for glyphosate resistance involving enzymatic conversion of glyphosate to N-acetylglyphosate (NAG) (1). Because the acetylated form of glyphosate has low affinity for the active site of EPSP synthase, it is nonherbicidal. Three variants of an enzyme that catalyzes N-acetylation of the secondary amine of glyphosate were discovered from Bacillus licheniformis. These GAT 5 enzymes are 17 kDa in size, are most active at pH 6.8, and have a K m for acetyl coenzyme A (AcCoA) of 1-2 M. The native enzymes are very poor catalysts for acetylation of glyphosate, with a k cat of 5.3 min Ϫ1 and K m,GPJ of 1.3 mM (for ST401 GAT, referred to hereafter as native GAT). 6 The B. licheniformis enzymes belong to a diverse family of largely uncharacterized bacterial N-acetyltransferases sharing between 30 and 64% amino acid identity to native GAT (Fig. 1). Despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (4).To develop gat as a transgene for glyphosate resistance in crops, we subjected the three B. licheniformis enzymes to 11 rounds of gene shuffling and obtained optimized variants with up to a 4,500-fold increase in catalytic efficiency (k cat /K m ) relative to the native enzyme. This was achieved through a combination of improvements in k cat (190-fold) and K m,GPJ (24-fold). When introduced into plants, optimized gat genes confer robust tolerance to glyphosate (1).To gain insight into the molecular mechanism of glyphosate N-acetylation by GAT, we determined the structure of a 7th round GAT variant (termed R7 GAT) in ternary complex with AcCoA and the competitive inhibitor 3-phosphoglycerate (3PG) and in binary complex with oxidized CoA. We also car-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2JDC, 2JDD) have been deposited in the Protein

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

“…The evolution of improved function appeared to occur through the accumulated effect of many small changes outside of the active site that influence the catalytic properties of the enzyme in a manner that is not obvious upon inspection of the structure. We reached a similar conclusion after evolving a microbial acetyltransferase for activity with glyphosate (Siehl et al, 2007). Although desensitization to herbicidal inhibitors was associated with a drop in k cat from approximately 200 min 21 to 70 to 80 min…”

Response of 98140 Corn With Gat4621 and hra Transgenes to Glyphosate and ALS-Inhibiting Herbicides

“…In addition, no transient yellowing was associated with the glyphosate application. These wholeplant results support previously published enzyme results that showed very high effectiveness of the GAT4621 metabolic system (Siehl et al 2007). …”

“…Recombinants with the highest activity then went through 11 iterative rounds of gene shuffling, a technique for molecular recombination of genes and directional screening to improve enzymatic activity (Ness et al 2002). At two points, sitedirected mutagenesis introduced additional amino acid substitutions to augment the 12 amino acid differences among the natural isozymes (Castle et al 2004;Siehl et al 2007). …”

PCR-Based Detection and Quantification of a Transgenic Glyphosate-Tolerant Canola Using a Novel Reference Gene System

“…Glyphosate tolerance, despite the occurrence of sporadic resistance in weeds (Powles 2008), remains a desirable trait, especially in combination with tolerance to herbicides with a different mode of action. A novel mode of glyphosatetolerant canola DP-Ø73496-4 has been developed by DuPont Pioneer, resulting from the incorporation of a glyphosate N-acetyltransferase (gat) transgene expressing an engineered acetylase capable of detoxifying the glyphosate herbicide (Castle et al 2004;Siehl et al 2005;Siehl et al 2007).…”