Biochemical analyses are instrumental in identifying the impact of mutations on holo and/or apo-forms and on the region(s) of alanine:glyoxylate aminotransferase variants associated with primary hyperoxaluria type I

Abstract

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations.

Fig. 1

3D representation of the AGT structure (PDB file 1H0C). (A) Overall structure of the AGT dimer. One monomer is colored gray, while in the opposite monomer the N-terminus, the large domain and the small domain are colored magenta, blue and green, respectively. PLP is represented as yellow sticks. Pro11 and Ile 340 are represented as red sticks, while Gly161, Ser187, Pro319 and Gly350 are represented as magenta sticks. (B) The AGT active site is shown. PLP is represented as yellow sticks. Residues directly interacting with PLP are represented as dark gray sticks or, if their mutation is analyzed in this study, as orange sticks. Ser218 is represented as magenta sticks. The dotted lines indicate possible hydrogen bond interactions. The figure was rendered using PyMol .

Fig. 2

CD spectra of AGT-Ma, AGT-Mi and pathogenic variants. (A) CD spectra of AGT-Ma (black), S158L-Ma (red), D183N-Ma (blue), S187F-Ma (violet), S218L-Ma (green) and P319L-Ma (orange).(B) CD spectra of AGT-Mi (black), W108R-Mi (green), G161C-Mi (fucsia), G161S-Mi (blue) and G350D-Mi (orange). All CD spectra are registered in the presence of saturating PLP concentrations in 100 mM potassium phosphate buffer, pH 7.4, at an enzyme concentration of 9 μM.

Fig. 3

Far-UV CD-monitored heating scans of AGT-Ma and AGT-Mi. Far-UV CD changes of holoAGT-Ma (black curve, straight line) and holoAGT-Mi (gray curve, straight line) in the presence of 10 μM exogenous PLP, and of apoAGT-Ma (black curve, dotted line) and apoAGT-Mi (gray curve, dotted line). The enzyme concentration was 10 μM, and the buffer was 100 mM potassium phosphate, pH 7.4.