Microbial preparation of nucleoside mono- and diphosphates

ANA L. VALINO, ROSARIO MÉDICI, ELIZABETH LEWKOWICZ, ADOLFO M. IRIBARREN,

Oviedo

Simposio; VIII Simposio Internacional de Biocatalisis y Biotransformaciones (BIOTRANS 2007); 2007

Universidad de oviedo

Nucleoside monophosphates (NMP) find several applications such as additives in the food industry and as therapeutic agents. For example, 5?-IMP and 5?-GMP have a characteristic taste and are used as flavour potentiators, and F-ara-AMP (9-β-D-rabinofuranosyl-2-fluoroadenine-5?-monophosphate) is a more water soluble and deaminase-resistant fluorinated analogue of arabinosyladenine that is currently used in the treatment of indolent B-cellmalignancies. To prepare NMP, chemical and enzymatic methods have been reported. Chemical phosphorylation involves multi-step, polluting and low yield processes. Employing enzymes,usually kinases, the syntheses are cleaner and moreefficient, but these enzymes are substrate specific and require ATP. More recently, non-specific acid phosphatases (NSAPs) from bacteria belonging to the Enterobacteriaceae familysuch as Morganella morganii, have been used to prepare 5?-IMP using cheaper phosphate donors. NSAPs from Shigella Flexneri was also employed in the regioselective phosphorylation of glucose and related compounds. Furthermore, the increasing demand of nucleosides triphosphates (NTP) as precursors of synthetic nucleic acids or as pharmacological drugs, makes necessary the development of biocatalysed strategies, as an alternative to the currently available chemical methods. The biosynthesis of these compounds involves the phosphorylation of NMPs to the corresponding diphosphates (NDPs) by nucleoside monophosphate kinases (NMPKs) and a second phosphorylation step from NDPs to NTPs by non-specific nucleoside diphosphate kinases. To carry out the first biotransformation bacterial NMPKs can be used, but they are high cost cofactors-dependent enzymes with narrow substrate specificity. In contrast, yeast NMPKs have broader specificity and acetone treated whole cells can be used to synthesise NDP without the need of cofactors addition. With the aim of preparing NMPs from the corresponding ribosides, we explored the NSAP activity of some Enterobacteria strains from our culture collection. The best yields of purine and pyrimidine ribonucleoside monophosphates were obtained using whole cells of Enterobacter aerogenes and Klebsiella planticola as biocatalysts. The biotransformation was also successfully applied to some deoxyribo- and arabinonucleosides. These NMPs were employed as starting materials to prepare NDPs using whole cell of Saccharomyces cerevisiae. In particular, to synthesise UMP and UDP another methodology, starting from the cheaper orotic acid, was carried out using whole cell of Brevibacterium ammoniagenes as biocatalyst.