Abstract
Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.
Highlights
Pendent enzyme of clinical relevance in that its deficiency is associated with primary hyperoxaluria type 1 (PH1), a rare genetic disease characterized by progressive renal failure due to accumulation of insoluble calcium oxalate [1]
Spectroscopic, and computational methods have demonstrated that (i) the enzyme is highly specific for catalyzing glyoxylate to glycine processing, (ii) pyridoxamine 5Ј-phosphate (PMP) remains bound to the enzyme during the catalytic cycle, and (iii) the AGT-PMP complex displays a reactivity toward keto acids higher than that of apoAGT in the presence of PMP [4]
The aim of this work was the study of the molecular basis of the functional synergism between the P11L and I340M polymorphisms and the F152I mutation giving rise to PH1 disease
Summary
Pendent enzyme of clinical relevance in that its deficiency is associated with primary hyperoxaluria type 1 (PH1), a rare genetic disease characterized by progressive renal failure due to accumulation of insoluble calcium oxalate [1]. AGT-Mi differs from AGT-Ma by two coding sequence polymorphisms (P11L and I340M) and a non-coding duplication in intron 1 These polymorphisms have no clinical significance on their own, but they enhance the deleterious effects of several common PH1 mutations that occur on the same allele [2]. One PLP cofactor is bound per subunit and is present in a Schiff base linkage to the active site lysine, Lys209 Analysis of this structure has allowed the rationalization of some, but not all, of the effects of disease-specific mutations [3]. PH1 patients carrying this mutation show the presence of mitochondrial AGT in addition to soluble peroxisomal AGT [6] The effect of this substitution on the minor allele cannot be understood in structural terms. Up to now the molecular defect of the F152I mutation associated with the minor allele (F152I-Mi) has not yet been identified
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