Document Detail


Reassessing the type I dehydroquinate dehydratase catalytic triad: kinetic and structural studies of Glu86 mutants.
MedLine Citation:
PMID:  23341204     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Samuel H Light; Wayne F Anderson; Arnon Lavie
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-02-11
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  22     ISSN:  1469-896X     ISO Abbreviation:  Protein Sci.     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-20     Completed Date:  2013-08-26     Revised Date:  2014-04-01    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  418-24     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 The Protein Society.
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MeSH Terms
Descriptor/Qualifier:
Glutamic Acid / genetics,  metabolism
Hydro-Lyases / chemistry*,  genetics,  metabolism*
Kinetics
Metabolic Networks and Pathways / genetics
Models, Molecular
Mutagenesis, Site-Directed
Salmonella enterica / enzymology,  genetics
Shikimic Acid / metabolism
Grant Support
ID/Acronym/Agency:
HHSN272200700058C//PHS HHS; HHSN272201200026C//PHS HHS
Chemical
Reg. No./Substance:
29MS2WI2NU/Shikimic Acid; 3KX376GY7L/Glutamic Acid; EC 4.2.1.-/Hydro-Lyases; EC 4.2.1.10/3-dehydroquinate dehydratase
Comments/Corrections

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