title: Study of the Phosphoryl-Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy.
abstract: The phosphoryl-transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D (1)H and (31)P NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ-phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure-based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymes.
authors.affiliation: Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Universidade de Santiago de Compostela, calle Jenaro de la Fuente s/n, 15782, Santiago de Compostela, Spain.
authors.forename: Verónica
authors.identifier: http://orcid.org/0000-0002-9812-7944
authors.initials: V
authors.lastname: Prado
authors.affiliation: Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Universidade de Santiago de Compostela, calle Jenaro de la Fuente s/n, 15782, Santiago de Compostela, Spain.
authors.forename: Emilio
authors.identifier: http://orcid.org/0000-0001-9489-9421
authors.initials: E
authors.lastname: Lence
authors.affiliation: Servicio de Microbioloxía-INIBIC, Complexo Hospitalario Universitario A Coruña (CHUAC), Hospital Teresa Herrera, As Xubias, 84, 15006 A, Coruña, Spain.
authors.forename: Juan A
authors.identifier: http://orcid.org/0000-0002-7581-8654
authors.initials: JA
authors.lastname: Vallejo
authors.affiliation: Servicio de Microbioloxía-INIBIC, Complexo Hospitalario Universitario A Coruña (CHUAC), Hospital Teresa Herrera, As Xubias, 84, 15006 A, Coruña, Spain.
authors.forename: Alejandro
authors.identifier: http://orcid.org/0000-0002-6340-7815
authors.initials: A
authors.lastname: Beceiro
authors.affiliation: Institute of Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Catherine Cookson Building, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Thompson
authors.affiliation: Institute of Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Catherine Cookson Building, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Alastair R
authors.initials: AR
authors.lastname: Hawkins
authors.affiliation: Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Universidade de Santiago de Compostela, calle Jenaro de la Fuente s/n, 15782, Santiago de Compostela, Spain. concepcion.gonzalez.bello@usc.es.
authors.forename: Concepción
authors.identifier: http://orcid.org/0000-0001-6439-553X
authors.initials: C
authors.lastname: González-Bello
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D016480:Helicobacter pylori / Q000737:chemistry / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology*; D010757:Phosphorous Acids / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010757:Phosphorous Acids; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; C043639:metaphosphoric acid; C002630:alpha,beta-methyleneadenosine 5'-triphosphate
keywords: drug discovery; enzyme catalysis; molecular dynamics; molecular modelling; reaction mechanism