On the Kinetic and Allosteric Regulatory Properties of the ADP-Glucose Pyrophosphorylase from Rhodococcus jostii: An Approach to Evaluate Glycogen Metabolism in Oleaginous Bacteria

Abstract

Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.

Glucosamine-6P and glucosamine-1P, respectively an activator and a substrate of rhodococcal ADP-glucose pyrophosphorylases, show a hint to ascertain (actino)bacterial glucosamine metabolism

“…From these results, it is tempting to speculate that GlcN-6P would represent a signal of high carbon/energy availability in rhodococci, as proposed for other ADP-GlcPPase activators in many bacteria and plants [7,8]. The hypothesis also agrees with hexose-6P (and its amino sugar related) representing a critical metabolic node and itself an allosteric regulator of the activity of actinobacterial ADP-GlcPPase [9,11], particularly in R. jostii [6].…”

“…We previously reported that Rjo ADP-GlcPPase distinctively uses GlcN-1P as a substrate [6]. Although such activity is one order of magnitude lower respect to the use of Glc-1P, it is relevant that for both substrates, Glc-6P exerts a significant allosteric activation of the enzyme activity.…”

“…Depending on the metabolite behaving as a regulator, ADP-GlcPPases were separated into nine groups [7], although such a classification is incomplete since it lacked data from Gram-positive organisms. We recently fill that gap by characterizing the enzyme from Actinobacteria (Gram-positive with high GC content DNA) and Firmicutes, finding distinctive allosteric effectors [6,9–12], which could add groups to that classification. Particularly, we established that glucose-6P (Glc-6P) is the primary activator in the ADP-GlcPPase from Actinobacteria and NADPH is a crucial inhibitor, thus connecting fatty acids synthesis with carbon to be allocated as glycogen [6,11].…”

“…We recently fill that gap by characterizing the enzyme from Actinobacteria (Gram-positive with high GC content DNA) and Firmicutes, finding distinctive allosteric effectors [6,9–12], which could add groups to that classification. Particularly, we established that glucose-6P (Glc-6P) is the primary activator in the ADP-GlcPPase from Actinobacteria and NADPH is a crucial inhibitor, thus connecting fatty acids synthesis with carbon to be allocated as glycogen [6,11]. The ADP-GlcPPases from Streptomyces coelicolor and R. jostii presented a broad sensitivity towards allosteric effectors according to organisms with broad metabolic capacities.…”

“…The ADP-GlcPPases from Streptomyces coelicolor and R. jostii presented a broad sensitivity towards allosteric effectors according to organisms with broad metabolic capacities. There, Glc-6P stands as a “leading activator”, exerting the highest activation ( S. coelicolor ) [11] or exhibiting the highest affinity ( R. jostii ) [6]. When the ADP-GlcPPase from R. jostii ( Rjo ADP-GlcPPase) was analyzed regarding its ability to use alternative sugar-1Ps, glucosamine-1P (GlcN-1P) was revealed as a substrate and, remarkably, was the only (besides Glc-1P) activity being activated by Glc-6P [6].…”