title: Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies.
abstract: Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.
authors.affiliation: Departamento de Química Bioorgánica, Instituto de Química Orgánica General, CSIC. Juan de la Cierva 3, Madrid 28006, Spain. isra.sanchez@iqog.csic.es.
authors.forename: Israel
authors.initials: I
authors.lastname: Sánchez-Moreno
authors.affiliation: Departament de Química Física i Analítica, Universitat Jaume I. Castellón 12071, Spain. bordes@uji.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Bordes
authors.affiliation: Departament de Química Física i Analítica, Universitat Jaume I. Castellón 12071, Spain. rcastill@uji.es.
authors.forename: Raquel
authors.initials: R
authors.lastname: Castillo
authors.affiliation: Departament de Química Física i Analítica, Universitat Jaume I. Castellón 12071, Spain. pernia@uji.es.
authors.forename: José Javier
authors.initials: JJ
authors.lastname: Ruiz-Pernía
authors.affiliation: Departament de Química Física i Analítica, Universitat Jaume I. Castellón 12071, Spain. moliner@uji.es.
authors.forename: Vicent
authors.initials: V
authors.lastname: Moliner
authors.affiliation: Departamento de Química Bioorgánica, Instituto de Química Orgánica General, CSIC. Juan de la Cierva 3, Madrid 28006, Spain. eduardo.junceda@csic.es.
authors.forename: Eduardo
authors.initials: E
authors.lastname: García-Junceda
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D015723:Gene Library; D008958:Models, Molecular; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011122:Polyphosphates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011122:Polyphosphates; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase
keywords: DHAP-dependent aldolases; biocatalysis; computational chemistry; dihydroxyacetone kinase; enzyme directed evolution; quantum mechanics/molecular mechanics