BIOCHEMICAL, FUNCTIONAL, AND REGULATORY CHARACTERIZATION OF THE FIRST KNOWN GLYCOGEN PHOSPHORYLASE INDEPENDENT OF THE PYRIDOXAL 5’-PHOSPHATE COFACTOR FROM THE METHANOGENIC ARCHAEON METHANOCOCCUS MARIPALUDIS.

The glycogen phosphorylase (GP) enzyme degrades the glycogen polymer into glucose-1-phosphate. All glycogen phosphorylases studied employ the pyridoxal-5'-phosphate (PLP) cofactor, bound to a strictly conserved lysine residue and essential for the phosphorolytic cleavage of glycogen. However, this cofactor is not present in the GP enzyme (MmGP) from the archaeon Methanococcus maripaludis, even though it displays glycogen phosphorylase activity. A multiple sequence alignment revealed that the strictly conserved lysine residue involved in PLP binding is replaced by threonine in all the GP enzymes from the order Methanococcales.  Moreover, scarce kinetic characterization or allosteric regulation has been described for GP enzymes from archaea despite the key role of this enzyme in sugar metabolism.

In this work, we characterize kinetically and spectroscopically the MmGP enzyme and its threonine mutants (T428A and T428K) to ascertain the significance of threonine in a PLP-independent catalytic mechanism and the ability of mutants to restore PLP cofactor binding. Also, the regulation of GP activity by several metabolic intermediates was assessed. MmGP exhibited a strong preference for long and branched oligosaccharides such as glycogen, with a KM of 0.3 mM for phosphate and a half-saturation constant of 0.3 mg/ml for glycogen, values similar to those reported for bacteria and eukarya. Fluorescence spectroscopy demonstrated the absence of the PLP cofactor in the wild-type enzyme. Interestingly, the PLP binding capacity was partially recovered in the T428K mutant but not in the T428A mutant. Kinetically, the T428A mutant shows kinetic parameters similar to the wild-type enzyme, whereas the T428K mutant has no significant enzyme activity. Lastly, wild-type MmGP was inhibited by several metabolites, including NaPPi, fructose-6P, PEP, ADP, ADP-glucose, and UDP-glucose, and was activated by fructose-1,6-bisphosphate (FBP), showing a different regulation pattern from the one reported for other GP enzymes.

This work was supported by FONDECYT Nº1231263