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.

FIGURE 1.

The substrate and product of the dehydration reaction catalyzed by DHQDs. Indicated is the numbering convention used throughout the text for the substrate, intermediate, and product and the pro-R and pro-S hydrogen relevant to the mechanism of elimination of the 1-hydroxyl group.

FIGURE 2.

Sequence and structure similarity between the cdDHQD and seDHQD type I DHQDs. A, sequence alignment of cdDHQD and seDHQD. Alignment was done in ClustalW2 version 2 using the default settings. Secondary features were defined using the cdDHQD structure in ESPript version 2.2. B, superposition of the biological dimer of the seDHQD pre-dehydration complex (cyan) and the cdDHQD post-dehydration complex (pink) structures (backbone root mean square deviation = 1.16 Å). Lys-170 and the reaction intermediates to which it is covalently bound are shown as sticks. Helices of the dimer interface are labeled.

FIGURE 3.

Crystal structures of DHQD in pre- and post-dehydration covalent intermediate states. A, structure of seDHQD active site with covalent pre-dehydration reaction intermediate. Carbon atoms are depicted in cyan, oxygens are in red, nitrogens are in blue, and sulfurs are in yellow. Difference maps were calculated with the reaction intermediate omitted from the model. The F_o − F_c map is contoured at the 3.0σ level (red), and the 2F_o − F_c map is contoured at the 1σ level (blue). An arrow indicates the 1-hydroxyl leaving group. B, schematic rendering of the pre-dehydration reaction intermediate shown in A. Distances between atoms are shown in angstroms. C, structure of the cdDHQD active site with covalent post-dehydration reaction intermediate. Model and maps are the same as A, except that carbons are shown in pink. The arrow points toward an ordered water molecule at a position consistent with that dehydrated from the substrate. D, schematic rendering of the post-dehydration reaction intermediate shown in C. Distances between atoms are shown in angstroms.

FIGURE 4.

The conformation of His-143 is dependent on the presence of the 1-hydroxyl group of the reaction intermediate. A, in the pre-dehydration reaction intermediate structure (cyan), the N^ϵ2 atom of His-143 is within interaction distance to the 1-hydroxyl (2.8 Å) and the pro-R C-2 hydrogen (2.7 Å). Thus, it can facilitate dehydration by transferring the hydrogen atom to the leaving hydroxyl group. B, superposition of the pre- (cyan) and post- (pink) dehydration states of DHQD. In the pre-dehydration state, His-143 interacts with the 1-hydroxyl group. Upon leaving of this group, as revealed by the post-dehydration intermediate structure, His-143 rotates to a position where it interacts with Glu-86 (2.9 Å).

FIGURE 5.

Proposed role of His-143 in type I DHQD-catalyzed reaction. A, putative role of His-143 in the catalytic dehydration. Following formation of the covalent Schiff base linker between Lys-170 and the substrate 3-dehydroquinate 1, His-143 assumes its pre-dehydration position, where its N^ϵ2 atom forms a key hydrogen-bonding interaction with the 1-hydroxyl group of the reaction intermediate. In this position, the His-143 N^ϵ2 atom abstracts the C-2 pro-R proton of the substrate to generate the carbanion intermediate 2, which gives rise to the enamine intermediate 3. The protonated His-143 then delivers its N^ϵ2 proton to facilitate departure of the 1-hydroxyl leaving group, which generates the ene-iminium intermediate 4. Because the H143 N^ϵ2 atom can no longer form the critical interaction with the 1-hydroxyl leaving group, a shift to the post-dehydration position of the residue ensues. Finally, following Schiff base hydrolysis, the formally dehydrated product 3-dehydroshikimate is released. Boxed intermediates 1 and 4 represent likely states of the reaction captured by pre- and post-dehydration crystal structures. B, role of His-143 in Schiff base formation and hydrolysis based on Leech et al. (25). In the formation of the Schiff base, attack by the Lys-170 N^ϵ atom on the 3-carbonyl carbon leads to formation of the carbinolamine intermediate. His-143 then delivers a proton to facilitate departure of the hydroxyl leaving group to generate the Schiff base intermediate. Following the catalytic hydrolysis described in A, His-143 adopts its post-dehydration conformation and catalyzes the reverse reaction to hydrolyze the Schiff base and regenerate the active site.

FIGURE 6.

Structure of the seDHQD K170M mutant with 3-dehydroquinate bound. A, active site of K170M mutant structure showing difference maps calculated and colored as described in the legend for Fig. 2. Each His-143 conformation is modeled at 50% occupancy. B, superposition of K170M substrate-bound (gray) and pre-dehydration reaction intermediate bound (cyan) structures. C, the two H143 side chain conformations are proximal to the 3-carbonyl oxygen of the 3-dehydroquinate. D, Lys-170 side chain modeled in to the active site of the K170M substrate-bound structure with the goal of maximizing the angle of approach of Lys-170 N^ϵ atom to the carbonyl carbon of the substrate (the angle defined by N^ϵ–C–O, i.e. the Bürgi-Dunitz angle).