Insights into the Mechanism of Type I Dehydroquinate Dehydratases from Structures of Reaction Intermediates

Abstract

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...

New insights into the mechanism of the Schiff base formation catalyzed by type I dehydroquinate dehydratase from S. enterica

“…It further means that the formation of the covalent Schiff base occurs until the substrate reaches the corresponding position in the crystal structure. This conclusion cannot support the hypothesis described above that the covalent Schiff base adduct may form before the substrate reaches its observed position in the crystal structure of the Schiff base intermediate (3M7W) [10]. This mode is special to type I DHQD by compared with several enzymes mentioned above that generate a similar Schiff base intermediate.…”

“…The results of several chemical modification and mutagenesis experiments clearly suggest that the Schiff base intermediate not only simply holds the substrate in the active site, but also plays important catalytic roles in the dehydration process [8,9]. This suggestion is also supported by the observed conformations in the recently reported crystal structures of type I DHQD from Salmonella enterica (PDB code: 3M7W and 3NNT) that the substrate adopts the similar position both in the Schiff base intermediate and in K170 M mutant with non-covalent complex [10]. However, the reaction mechanism of the Schiff base formation is not very clear so far.…”

“…The initial structure of the substrate is from the crystal structure of K170 M mutant of type I DHQD from S. enterica with non-covalent complex with dehydroquinate (PDB code: 3NNT) [10], and the initial structure of the wild type enzyme is generated on the basis of the same crystal structure through replacing Met170 by Lysine. The ionization states of His143 and Lys170 were set on the basis of the experimental studies [9,[15][16][17].…”

“…According to the stereoelectronic principles, it is possible for the highly conserved Lys170 residue to approach the 3-carbonyl carbon (C 3 ) atom of the substrate at the Bürgi-Dunitz angle of *107° [11] and form the covalent Schiff base between them. However, modeling Lys170 to the crystal structure of K170 M mutant in non-covalent complex shows that the maximal approach angle of the NZ atom in Lys170, the C 3 atom in the substrate, and the 3-carbonyl oxygen (O 3 ) atom in the substrate (NZ-C 3 -O 3 ) is 58° [10], which is much less than the Bürgi-Dunitz angle. Thus, the covalent Schiff base may form either by a non-Bürgi-Dunitz approach with the substrate in its observed position or by a Bürgi-Dunitz approach with the substrate in its unobserved position in the active site.…”

“…Thus, the covalent Schiff base may form either by a non-Bürgi-Dunitz approach with the substrate in its observed position or by a Bürgi-Dunitz approach with the substrate in its unobserved position in the active site. In the latter case, during the process of the substrate docking into the active site, the substrate reaches a first binding position where the Bürgi-Dunitz angle requirement is met and the covalent Schiff base is formed and then reorganizes its position to be the observed one in the crystal structure of type I DHQD from S. enterica with the Schiff base intermediate [10]. In fact, this suggested binding mode is found in the Schiff base formation catalyzed by several other enzymes, such as fructose 1,6-bisphosphate aldolase [12], dihydrodipicolinate synthase [13], and 2-keto-3-deoxy-6-phosphogluconate aldolase [14].…”