Reassessing the type I dehydroquinate dehydratase catalytic triad: kinetic and structural studies of Glu86 mutants

Abstract

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined-Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction-the role of Glu86 remains poorly characterized. To probe Glu86's role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild-type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.

Figure 1

(A) Superposition of seDHQD unliganded (pink, PDB code: 3L2I) and bound DHQ reaction intermediate (gray, PDB code: 3M7W) structures, displaying the putative catalytic triad: Glu86, His143, and Lys170. Distances are shown in angstroms and colored by structure. (B) Maximal DHQD activity by pH of wild-type, E86Q, and E86A variants. Activity is normalized to the most active pH for each variant. Standard deviations are indicated as error bars.

Figure 2

Three modes of DHS binding from two E86A mutant crystal structures. (A) Stick model of the first protomer of the cocrystallized E86A-DHS complex. The product, DHS, is colored yellow in all panels. Select active site residues are colored cyan. Throughout the figure, fo-fc electron density maps (red) calculated with ligand omitted from the model are contoured at 2.5σ. (B) Superposition of the first protomer of the cocrystallized E86A-DHS complex (cyan) to the K170M-DHQ complex (beige, PDB code: 3NNT). DHQ is colored brown. (C) Stick model of second protomer of the cocrystallized E86A-DHS complex. (D) Superposition of the second protomer of the cocrystallized E86A-DHS complex to wild-type the Schiff base bound reaction intermediate structure (light gray, PDB code: 3M7W). The ordered water molecule (W1) in the E86A-DHS structure situated in the position vacated by the His143 imidazole is represented as a cyan sphere. A dashed line traces disordered residues Lys85-Gly88. (E) Stick model of the soaked E86A-DHS complex (green). (F) Superposition of the soaked E86A-DHS complex (green) to the wild-type Schiff base bound reaction intermediate (light gray). DHQ is colored dark gray. The ordered water molecule (W2) in the E86A-DHS structure situated in the position vacated by the Glu86 side chain is represented as a green sphere.