title: 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.
authors: Center for Structural Genomics of Infectious Diseases and Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
authors: Samuel H
authors: SH
authors: Light
authors: Wayne F
authors: WF
authors: Anderson
authors: Arnon
authors: A
authors: Lavie
mesh_terms: D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019779:Salmonella enterica / Q000201:enzymology / Q000235:genetics; D012765:Shikimic Acid / Q000378:metabolism
chemical_list: D012765:Shikimic Acid; D018698:Glutamic Acid; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase