On the Role of Genes Coding for Sucrose Synthase in Anabaena variabilis

M.A. PUNTILLO; M.D. ASENCIÓN DIEZ; A.A. IGLESIAS; M.A. BALLICORA

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

SAIB

Sucrose (Suc) is mainly found in organisms performing oxygenic photosynthesis. The disaccharide plays a central physiological role for carbon metabolism, nitrogen fixation and stress tolerance in filamentous cyanobacteria. It has been described that plant Suc synthase (SucSase; EC 2.4.1.13) is mainly involved in catalyzing the reversible conversion of Suc and UDP into fructose (Fru) and UDP-glucose (UDPGlc). Anabaena variabilis has two genes (susA and susB) respectively encoding for forms A and B of SucSase. These genes were synthesized de novo for optimal expression in Escherichia coli and cloned in pET28c vector to produce active SucSaseA and SucSaseB purified to homogeneity. To catalyze synthesis of Suc, SucSaseA showed 3-fold higher affinity towards ADPGlc (S0.5 = 0.8 mM) than for UDPGlc, with similar values of Vmax. The S0.5 for Fru (3.6 mM) was 33-fold lower when using ADPGlc compared with UDPGlc as the co-substrate. SucSaseB mutated in key amino acid residues exhibited 2.5 mU/mg of activity (100-fold lower than that of SucSasaA) when assayed with 20 mM ADPGlc and 40 mM Fru. No activity was observed with UDPGlc (up to 400 mM). Genes susA and susB were subcloned in vectors allowing coexpression (pDUET) to explore for the production of heterooligomeric forms of the enzyme. Results contribute to a better understanding on the occurrence, regulation and evolution of Suc metabolism.