Dihidroxiacetona quinasa de Citrobacter freundii CECT 4626 una aproximación integral a un nuevo biocatalizador para la formación de enlaces C-C

Resum

Aldolases have been extensively used in chemoenzymatic syntheses because of their ability to catalyze the formation of C-C bonds with a high degree of stereochemical control. The main group of aldolases from the biocatalytic point of view is the one that uses dihydroxyacetone (DHA) phosphate as donor. Their main drawback is their strict specificity for the donor substrate. A straightforward strategy for DHAP preparation is the kinasecatalysed DHA phosphorylation, using ATP as phosphoryl donor. From this point of view, ATP-dependent DHA kinases have been given considerable attention because their feasibility for the simple and efficient obtaining of DHAP. In this Thesis, an integral study of DHAK from Citrobacter freundii CECT 4626 as biocatalyst is described. This study includes: i) The cloning over-expression and a complete biochemical characterization of the enzyme. In the context of this study we also describe the promiscuous behaviour of this ATP-dependent DHAK. This enzyme, beside the transfer of the ¿-phosphate of the ATP to DHA is able to catalyse the cyclization of the FAD to yield riboflavin 4¿,5¿-cyclic phosphate (cFMN). ii) A multi-enzyme system for one-pot C-C bond formation, based on the use of this DHAK for in situ DHAP formation and DHAP-dependent aldolases for the catalysis of the aldol reaction, has been designed and developed. The multienzyme system was completed with the in situ regeneration of ATP catalysed by AK. In the context of this multi-enzyme system we have described for the first time that an ¿,ß-unsaturated aldehyde is efficiently accepted as substrate by RAMA. iii) Finally, we have initiated a Directed evolution Program to modified the specificity of DHAK for the phosphoryl donor. We have performed a first evolution generation consisting in a mutagenesis round followed by a round of artificial recombination. Surprisingly, when we screened this first generation for DHAK activity using poly-P as phosphoryl donor, we found sixteen positive mutants. After purification of these sixteen mutants, we could prove that three of them had activity in the order of 10 mU per mg of protein.