The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre-and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.Present in bacteria, fungi, and plants but absent in higher eukaryotes, the seven enzymes of the shikimate pathway catalyze sequential reactions to generate chorismate. Chorismate serves as a precursor of many biologically important aromatic compounds including the ubiquinones, folates, and aromatic amino acids (1). The essential nature of these proteins in a number of species, in combination with an absence of human homologs, makes the shikimate pathway an attractive target for the development of non-toxic antimicrobials, anti-fungals, and herbicides (2-4). Given the complexities inherent to inhibitor design, a comprehensive understanding of the structural and mechanistic framework underlying the function of the shikimate pathway enzymes should aid in the development of novel shikimate-targeting inhibitors.The third step in the shikimate pathway, consisting of the dehydration of dehydroquinate to dehydroshikimate (Fig. 1), can be catalyzed by two unrelated enzymes, termed type I and type II dehydroquinate dehydratases (DHQDs).2 These two enzyme families lack sequence or structural homology and employ distinct reaction mechanisms (5-10). The type I DHQDs utilize a covalent Schiff base (imine) intermediate that results in a cis-elimination, whereas the type II reaction lacks a covalent intermediate and undergoes a trans-elimination (5-10). The phylogenetic distribution of the two enzyme types is somewhat disorderly, with closely related s...