On the Regulatory Properties of the Rhodococcus jostii ADP-glucose Pyrophosphorylase

A.E. CEREIJO; M.D. ASENCIÓN DIEZ; A.A. IGLESIAS; H.M. ÁLVAREZ

Rosario

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

SAIB

Rhodococcus jostii is an actinobacterium (Gram-positive, GC rich) accumulating lipids as a main carbon and energy storage, although it also produces glycogen as a temporal reserve molecule. To shed light into relationships between the polyglucan synthesis and lipids metabolism, we performed the molecular cloning of the glgC gene, encoding ADP-glucose pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) from R. jostii. The gene was heterologously coexpressed with GroEL/GroES in Escherichia coli BL21 (DE3) to achieve the production of recombinant R. jostii ADP-Glc PPase with high purity level. The purified enzyme depicted a Vmax of 0.39 U/mg in the ADP-Glc synthesis direction, with S0.5 values for both substrates around 1 mM and exhibiting sensitivity to allosteric regulation bymetabolites. Glucose-6P, fructose-6P, mannose-6P, ribose-5P and PEP are activators enhancing Vmax and the affinity of the enzyme towards Glc-1P; whereas NADPH, pyruvate and 6P-gluconic acid behaved as main inhibitors. Analysis on the interplay between the different effector molecules suggest a fine tuning of the R. jostii ADP-Glc PPase activity in a metabolic frame relating in an opposite form the pathways for glycogen and lipids synthesis. Results support a model where allosteric regulation of ADP-Glc PPase is critical for determining a role of glycogen as a temporal carbon storage molecule in R. jostii.