Abstract
Glycogen phosphorylase (GP) is a critical enzyme in glycogen metabolism. Even though methanogens from the archaeal orders Methanosarcinales and Methanococcales are unable to grow on sugars, they store glycogen, which is metabolized through the glycogenolysis and glycolytic pathways when the carbon source for methanogenesis is depleted. Under these metabolic conditions, the activity of the GP enzyme is essential. To be active, all phosphorylases characterized to date require the cofactor pyridoxal 5’-phosphate (PLP). This cofactor is covalently bound via Schiff base to a strictly conserved lysine residue at the active site. Extensive GP sequence analysis of organisms from different domains of life shows strict conservation of active site residues despite significant differences in sequence length. Interestingly, in GP sequences of organisms from the order Methanococcales of archaea, a threonine residue replaces the conserved lysine involved in PLP binding. The purification and characterization of recombinant GP from Methanococcus maripaludis show that the enzyme exhibits glycogen phosphorylase activity and high specificity 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. The results demonstrate that an archaeal GP from the order Methanococcales performs catalysis without the PLP cofactor, deviating from the well-established phosphorylase catalytic mechanism and revealing new scenarios for the glucosyltransferase reaction. Moreover, analysis of enzyme regulation shows that the activity is affected by various molecules, including nucleotides, intermediates of carbon metabolism, and phosphate species. Most of these molecules have not previously been identified as regulators of glycogen phosphorylases in prokaryotes. These results suggest that other GPs from Methanococcales can undergo complex regulation.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- AC50
- half-maximal Activitory concentration
- ADP
- Adenosine 5’-diphosphate
- ADPG
- Adenosine-5’-diphosphoglucose
- AMP
- Adenosine 5’-monophosphate
- ATP
- Adenosine 5’-triphosphate
- BG
- Bovine glycogen
- CMP
- Cytidine 5’-monophosphate
- FBP
- Fructose 1,6-bisphosphate
- G1P
- Glucose 1-phosphate
- GMP
- Guanosine monophosphate
- IC50
- half-maximal inhibitory concentration
- M3
- Maltotriose
- M4
- Maltotetraose
- M5
- Maltopentaose
- MD1
- Maltodextrin dextrose equivalent 4-7 unit
- MD2
- Maltodextrin dextrose equivalent 8-15 unit
- MmGP
- Glycogen phosphorylase from Methanococcus maripaludis
- NaPPi
- Tetrasodium pyrophosphate
- OG
- Oyster glycogen
- OcGP
- Glycogen phosphorylase from Oryctolagus cuniculus
- PEP
- Phosphoenolpyruvate
- PLP
- Pyridoxal-5’-phosphate
- PYR
- Pyruvate
- UDPG
- Uridine 5’-diphosphate glucose