The availability of high-resolution crystal structures of PNPO in a complex with PLP and the cofactor FMN (PDB accession number 1nrg, allows assessing the molecular caused by the reported variants. Based on their position in the protein structure and type of mutation, the mutations of PNPO can be separated into four categories. The largest category (here termed category I) contains mutations that directly affect the catalytic site and its capacity for ligand and cofactor binding. These mutations include R95C, R116Q, R141C, R161C, R225C/L/H, and R229W/Q. Strikingly, all these substitutions change a long and positively charged arginine into a polar or hydrophobic residue with different side chain dimensions. Accordingly, all of them impair the coordination of the negatively charged phosphate moieties of PLP or FMN, and sometimes have additional effects (e.g. loss of π-stacking interactions with PLP in the case of R225 mutations, or loss of inter-chain ionic bonds for R116Q).
The second category, category II, contains mutations that affect the fold and stability of the protein because of a nonconservative side chain substitution. Their effects range from mild loss of stabilizing surface interactions (E50K) and loss of hydrophobic interactions (P213S) to severely destabilizing clashes (G118Q/R).
A third category (category III) is constituted by variants causing residue deletion (S93S_A94Ldel), premature stop codons (A174X), loss of stop codons (X262Q) and frameshifts combined with loss of subsequent protein sequences (L83W_fs*17, P150R_fs*27). In the compact structure of the PNPO dimer a significant loss of residues would severely compromise the protein’s structure and function. Hence, the truncating mutations are expected to lead to nonfunctional proteins. PNPO has the particularity that its C-terminus folds back inside the protein and terminates just before the substrate-binding pocket. Accordingly, the loss of stop codon would disrupt both, ligand binding and protein fold.
D33V is in a human-specific N-terminal extension and does not affect the 3D structure or catalytic function of the protein, placing it in category IV. D33V might mildly affect the (unknown) function of the N-terminal extension.
PNPO