title: Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases.
abstract: DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymes.
authors.affiliation: *Departamento de Ciencias Biomédicas, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: Claire
authors.initials: C
authors.lastname: Coderch
authors.affiliation: †Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Lence
authors.affiliation: †Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Peón
authors.affiliation: ‡Institute of Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, U.K.
authors.forename: Heather
authors.initials: H
authors.lastname: Lamb
authors.affiliation: ‡Institute of Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, U.K.
authors.forename: Alastair R
authors.initials: AR
authors.lastname: Hawkins
authors.affiliation: *Departamento de Ciencias Biomédicas, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: Federico
authors.initials: F
authors.lastname: Gago
authors.affiliation: †Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Concepción
authors.initials: C
authors.lastname: González-Bello
mesh_terms: D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D016480:Helicobacter pylori / Q000201:enzymology*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011789:Quantum Theory; D012997:Solvents
chemical_list: D001426:Bacterial Proteins; D012997:Solvents; D001224:Aspartic Acid; D001120:Arginine; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase