Glycogen phosphorylase from the methanogenic archaeon Methanococcus maripaludis: unique regulatory properties of a pyridoxal 5'-phosphate independent phosphorylase.

Abstract

Methanogens from the archaeal orders Methanosarcinales and Methanococcales cannot grow on sugars. However, they store glycogen, which is metabolized through the glycogenolysis and glycolytic pathways when carbon sources for methanogenesis are depleted, with the activity of glycogen phosphorylase (GP) being essential for this process. All phosphorylases characterized to date require the cofactor pyridoxal 5'-phosphate (PLP) covalently bound via a Schiff base to a strictly conserved lysine residue at the active site for their activity. Extensive GP sequence analysis of organisms from different domains of life shows strict conservation of active site residues despite significant differences in sequence length. In GP sequences of organisms from the order Methanococcales of archaea, a threonine residue replaces the conserved lysine involved in PLP binding. Characterization of recombinant GP from Methanococcus maripaludis demonstrated that the enzyme had GP activity and great selectivity for glycogen as a substrate. Analysis of the PLP content performed by several methods, such as absorbance, fluorescence, cyanohydrin adduct formation, and mass spectrometry, confirmed the absence of PLP. These results represent a unique case of a GP being active without the PLP cofactor, offering new insights into the glucosyltransferase reaction mechanism. Moreover, analysis of enzyme regulation shows that the activity is affected by various molecules, including nucleotides, intermediates of carbon metabolism, and phosphate species. The regulation of this archaeal GP by multiple metabolic intermediates suggests a role for this regulation in the organism's adaptability to environmental and nutritional changes, suggesting that it can be shared with GPs from other methanogenic species.