A conserved surface loop in type I dehydroquinate dehydratases positions an active site arginine and functions in substrate binding

Abstract

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

Figure 1

Reaction catalyzed by DHQD.

Figure 2

Dynamic loop behaviour in DHQD. Overlay of open loop-P2₁2₁2₁ (green) and pre-dehydration intermediate bound (cyan, PDB code 3M7W) DHQD structures (RMSD = 0.28 Å over 198 Cα atoms, calculated with Val228 to Gln236 residues omitted). A dotted green line traces where the disordered loop residues may lie. Inset highlights differential loop behaviour between the two structures and shows interaction of closed loop residues with the reaction intermediate (shown in pink).

Figure 3

The closed loop conformation in the closed loop-P2₁ structure likely results from crystal packing. (a) Superposition of pre-dehydration intermediate bound (cyan) and closed loop-P2₁ (yellow) structures. The reaction intermediate is shown in pink. (b) Superposition of the closed loop-P2₁2₁2₁ (yellow) and open loop-P2₁2₁2₁ (green) structures. Residues His51 and Glu89 of a symmetry related molecule are proximal to the closed loop and would clash with an open loop conformation. A dotted green line traces where the disordered residues may lie.

Figure 4

Chloride binding within the closed loop-P2₁ active site. (a) Superposition of closed loop- P2₁ (yellow) and pre-dehydration reaction intermediate bound (cyan) structures. The chloride and 1-carboxyl group localize to the same position within the active site. The reaction intermediate is shown in pink. (b) A schematic representation of the closed loop-P2₁ active site shows key chloride interactions.

Figure 5

Loop closure results in a conformational change of Arg213. (a) Sequence alignment of the type I DHQDs from S. enterica, C. difficile, E. faecalis, and A. thaliana done in ClustalW2 version 2 using ESPript version 2.2. Asterisks denote S. enterica Ser232 and Gln236, which are observed to hydrogen bond with the reaction intermediate. (b) Superposition of open loop-P2₁2₁2₁ (green) and pre-dehydration reaction intermediate bound (cyan) active sites. The reaction intermediate bound conformation of Arg213 positions the residue to form a bidentate salt bridge (yellow dashes) with the reaction intermediate’s (shown in pink) 1-carboxyl group. Distances that would be unusually short between the open loop-P2₁2₁2₁ conformation of Arg213 and reaction intermediate bound conformation of Gln236 (red dashes) are shown in angstroms. Arrows denote the direction of Gln236 and Arg213 movement as the loop transitions from open to closed states. (c) Superposition of pre-dehydration reaction intermediate (cyan) and closed loop- P2₁ (yellow) structures. Arg213 adopts identical conformations in each structure.

Figure 6

The Q236A variant enzyme fails to induce a conformational change of Arg213 and has altered anion coordinating properties. (a) Superposition of open loop-P2₁2₁2₁ (green), pre-dehydration reaction intermediate bound (cyan), closed loop-P2₁ (yellow), and closed loop-Q236A (magenta) active sites. The reaction intermediate is shown in pink. In both closed loop WT structures Arg213 adopts its ligand bound conformation. In the Q236A variant structure Arg213 remains in its apo conformation. (b) Superposition of the closed loop-P2₁ (yellow) and closed loop-Q236A (magenta) active sites. Corresponding to the altered Arg213 conformation, the chloride is displaced 3.0 Å within the Q236A active site relative to its position in the WT enzyme.