Aplicación de una fosfopentomutasa sobreexpresada en la síntesis de adenosina a partir de ribosa-5-fosfato

TAVERNA PORRO, M. L.; BOUVIER, L.; PEREIRA, C.; MONTSERRAT, J.M.; IRIBARREN, A.

San Pablo

Congreso; Biocatálisis y biotransformaciones 2006. Segundo encuentro regional.; 2006

Preparation of modified nucleosides is a theme of great importance, due to the multiple applications of these molecules. They had a great impact in pharmaceutical chemistry since almost all the antivirals, nowadays, are nucleoside analogs. (for example: AZT, ddC, ddI, d4T, against HIV virus and acyclovir, gancyclovir, against Herpes infection). On the other hand, these molecules have had also repercussion in the chemistry of modified nucleic acids. These analogs, permitted to incorporate chemical modifications in the structure of RNA and DNA, conferring improved enzymatic and chemical stability. Biotechnological synthesis of nucleosides presents some advantages over traditional chemical methods. Usually the reaction conditions are milder and environmentally cleaner and the regio and estereoselectivitiy of the process is higher. A biotechnological method consists in a transglycosilation reaction using a pyrimidine nucleoside and a purine base. However, an important limitation of this strategy is the need of the corresponding modified pyrimidine nucleoside. Since ribose-1-phosphate is an intermediate of the transglycosilation reaction, therefore it can be directly use as the sugar source. The disadvantage of this approximation is given by the difficult preparation of ribose-1-phosphate and by its poor stability under reaction conditions. These disadvantages could be surprassed by using ribose-5-phosphate as the sugar donor, coupled with phosphopentomutase activity. This enzyme catalyses the steroespecific migration of 5-phosphate-α-1 position of ribose. With this aim, phosphopentomutase of Escherichia Coli was expressed, transforming the strain of E. Coli BL21, with pRSet-ppm plasmid (cloned in our laboratory). Growth and induction times, were optimized, as well as enzyme purification. Activity experiments were made, modifying the substrate and cofactors concentration and pH, with the aim of improving the kinetic and reaction yield. Preliminary results afforded 50% yield in 20 hours, and further optimization is being carried out. Finally we are exploring the possibility of preparing nucleosides analogs, using this enzymatic route.