PNGases : a diverse family of enzymes related by function rather than catalytic mechanism : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular BioSciences, Massey University, Palmerston North
Peptide:N-glycanases (PNGases, EC 188.8.131.52) release N-linked glycan moieties from glycoproteins and glycopeptides. They catalyse the cleavage of the amide bond between the proximal N-acetylglucosamine and the asparagine side chain of the polypeptide, resulting in the conversion of the asparagine residue to aspartic acid and the concomitant release of the intact glycan and free ammonia. PNGases, especially PNGase F, are valuable tools for the removal of glycan moieties from glycoproteins for subsequent analyses of the released glycan and/or protein.
In the first part of this work, a classification for PNGases has been proposed, dividing these enzymes into three types based on their primary amino acid sequence, and also on their subcellular localisation, phylogenetic distribution (to date) and physiological function (if known). It appears that the three PNGase-types developed by convergent evolution. Gene expression studies for one putative type I (Deinococcus radiodurans) and two putative type II (Aspergillus niger, Streptomyces avermitilis) PNGases showed that these proteins were expressed in their native organisms. Recombinant expression of these proteins and the putative PNGase from Sulfolobus solfataricus yielded soluble protein for the S. avermitilis and D. radiodurans proteins and PNGase activity could be shown once for the latter enzyme.
In the second part of this work, site-specific mutants of PNGase F, the only characterised type I PNGase to date, were generated, expressed and characterised using enzyme kinetic methods. From the kinetic results obtained here, a catalytic mechanism can be proposed for PNGase F. In this mechanism a bound water molecule acts as the nucleophile after being activated by the abstraction of a proton by a conserved glutamate residue. The carbonyl carbon of the scissile bond is primed for the nucleophilic attack by another conserved residue, Arg248, probably by the donation of a proton.
A 1.57 Å crystal structure of the recombinant wildtype PNGase F that has three glycerol molecules non-covalently bound in the active site is also presented. This crystallographic analysis shows that the recombinant protein has a structure identical to that of the native protein, validating the basis of the kinetic studies, and showing why glycerol acts as an inhibitor of this enzyme.