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.

Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

“…We compared the growth of yeast expressing mutant forms of AGT with their in vitro melting temperatures calculated in the holo-(bound to a PLP cofactor) or apo-forms, shown in Table 1. Previous work has shown that the apo-form of AGT unfolds in two steps indicative of two domains, which can be represented by two melting temperatures, T m1 and T m2 (Oppici et al 2012). Several mutant AGT variants show no defect in T m1 , but do not undergo a T m2 transition, indicative of a molecular defect confined to the second domain (Oppici et al 2012).…”

“…Previous work has shown that the apo-form of AGT unfolds in two steps indicative of two domains, which can be represented by two melting temperatures, T m1 and T m2 (Oppici et al 2012). Several mutant AGT variants show no defect in T m1 , but do not undergo a T m2 transition, indicative of a molecular defect confined to the second domain (Oppici et al 2012). In addition to alleles analyzed in Figure 1, we examined S158L-ma, W108R-mi, G350D-mi, and G161S-mi.…”

“…In addition to alleles analyzed in Figure 1, we examined S158L-ma, W108R-mi, G350D-mi, and G161S-mi. The mutations S158L and W108R are in residues that contact the PLP cofactor in the active site of the enzyme (Zhang et al 2003), and these mutants show significantly reduced catalytic activity and reduced affinity for PLP (Oppici et al 2012). The G161S mutation is in a region bridging the two domains of AGT, and both holo-and apo-forms are destabilized.…”

“…The G161S mutation is in a region bridging the two domains of AGT, and both holo-and apo-forms are destabilized. G350D, in a random coil region near the active site, has been proposed to specifically affect the structure of the second domain, as measured by a loss of T m2 (Oppici et al 2012).…”

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview

“…Expression in mammalian cells is considered as the closest model of in vivo conditions and gives information on the expression level, intracellular stability and targeting of the AGT variants [17,28,35,40,[46][47][48][49][50]. On the other hand, the purification and characterization of the variants expressed in E. coli are suitable to dissect and quantify the effect of a mutation on the functional properties of AGT as well as on its secondary, tertiary and quaternary structures [11][12][13]17,18,35,36,[51][52][53][54][55][56][57][58]. Yeast and cell-free systems allow a rapid but semi-quantitative determination of the impact of each mutation on the intracellular activity, stability and assembly of the protein [37,53,54,59].…”