title: Preparation and characterization of a bifunctional aldolase/kinase enzyme: a more efficient biocatalyst for C-C bond formation.
abstract: A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening linker of five amino acid residues. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structures of the bifunctional enzyme and the parental enzymes were analyzed by circular dichroism (CD) spectroscopy to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Because S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centers in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channeling.
authors.affiliation: Departamento de Química Bioorgánica, Instituto de Química Orgánica General, CSIC, Juan de la Cierva 3, 28006, Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Iturrate
authors.forename: Israel
authors.initials: I
authors.lastname: Sánchez-Moreno
authors.forename: Isabel
authors.initials: I
authors.lastname: Oroz-Guinea
authors.forename: Jesús
authors.initials: J
authors.lastname: Pérez-Gil
authors.forename: Eduardo
authors.initials: E
authors.lastname: García-Junceda
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D013210:Staphylococcus / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: D010770:Phosphotransferases; D000446:Aldehyde-Lyases