Natural and modified versions of the 5-enolpyruvylshikimate-3-phosphate synthase () gene have been used to confer tolerance to the broad-spectrum herbicide glyphosate in a variety of commercial crops. The most widely utilized trait was obtained from the strain CP4 and has been commercialized in several glyphosate-tolerant crops. The EPSPS gene products are enzymes that have been divided into three classes based on sequence similarity, sensitivity to glyphosate, and steady-state catalytic parameters. Herein, we describe the informatics-guided identification and biochemical and structural characterization of a novel EPSPS from (DGT-28 EPSPS). The data suggest DGT-28 EPSPS and other closely related homologues exemplify a distinct new class (Class IV) of EPSPS enzymes that display intrinsic tolerance to high concentrations of glyphosate ( ≥ 5000 μM). We further demonstrate that , when transformed into stable plants, provides robust (≥4× field rates) vegetative/reproductive herbicide tolerance and has utility in weed-control systems comparable to that of commercialized events.