Closing down on glyphosate inhibition--with a new structure for drug discovery.

A enzyme inhibitors used in agriculture, glyphosate (N-phosphomethyl glycine) is remarkable. A nonselective herbicide discovered in 1970 by a group of scientists at Monsanto led by Dr. John Franz (1), glyphosate, since first commercialization under the trade name Roundup, has been used globally as a safe and effective means of weed control. The discovery of glyphosate’s herbicidal activity was not quite serendipity, but instead resulted from a synthetic strategy based on the hypothesis that the weak herbicidal activities of related compounds derived from the possibility of their similar metabolic fate (2). Nevertheless, the initial discovery by greenhouse screening has been followed by intensive biochemical studies that have now led to nearly complete understanding of glyphosate’s mode of action. In 1972, scientists at Monsanto led by Dr. E. Jaworski observed (3) that application of glyphosate resulted in the inhibition of aromatic amino acid biosynthesis in plants. In 1980, Professor N. Amrhein and coworkers (4) identified its target enzyme from the shikimate pathway (4): 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19). EPSPS is a key enzyme involved in aromatic amino acid biosynthesis (5). The enzyme catalyzes an unusual reaction, wherein the enolpyruvoyl group from phosphoenol pyruvate (PEP) is transferred to the 5-hydroxyl of shikimate3-phosphate (S3P) to form the products 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate (Pi). The only other enzyme known to catalyze carboxyvinyl transfer by using PEP is UDP-Nacetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step in the biosynthesis of the peptidoglycan layer of the bacterial cell. In the case of EPSPS, the reaction proceeds through a tetrahedral intermediate (Scheme 1) formed from S3P and PEP (6). Inhibition of EPSPS by glyphosate has been shown to proceed through the formation of an EPSPS-S3P-glyphosate ternary complex and the binding is ordered with glyphosate binding to the enzyme only after the formation of a binary EPSPS-S3P complex. Binding of glyphosate to EPSPS has been shown to be competitive with PEP and uncompetitive with respect to S3P (7). What makes glyphosate a remarkable inhibitor and herbicide? Glyphosate is a relatively simple molecule—an N-methyl phosphonate derivative of glycine with a chemical structure not unlike that of the universal high energy phosphoryl-transfer agent PEP. Despite this, glyphosate retains exquisite specificity for EPSPS and is not known to appreciatively inhibit any other enzyme, even MurA (8). The EPSPS reaction is the penultimate step in the shikimic acid pathway for the biosynthesis of aromatic amino acids (Phe, Tyr, and Trp) and many secondary metabolites, including tetrahydrofolate, ubiquinone, and vitamin K (9). The shikimic acid pathway, present in plants and microorganisms, is completely absent in mammals, fish, birds, reptiles, and insects. The importance of the shikimate pathway in plants is further substantiated by the estimation that up to 35% or more of the ultimate plant mass in dry weight is represented by aromatic molecules derived from the shikimate pathway (2). On the basis of this information, it is readily apparent why EPSPS is a good target for novel antibiotics and herbicides. The crystal structure of the ternary EPSPS-S3P-glyphosate complex was reported in PNAS at a stunning resolution of 1.5 Å and allows detailed visualization of the complete set of interactions of the