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  • Title: Effects of interface mutations on the dimerization of alanine glyoxylate aminotransferase and implications in the mistargeting of the pathogenic variants F152I and I244T.
    Author: Dindo M, Montioli R, Busato M, Giorgetti A, Cellini B, Borri Voltattorni C.
    Journal: Biochimie; 2016 Dec; 131():137-148. PubMed ID: 27720751.
    Abstract:
    In this work the dimerization process of the minor allelic form of human alanine glyoxylate aminotransferase, a pyridoxal 5'-phosphate enzyme, was investigated. Bioinformatic analyses followed by site-directed mutagenesis, size exclusion chromatography and catalytic activity experiments allowed us to identify Arg118, Phe238 and Phe240 as interfacial residues not essential for transaminase activity but important for dimer-monomer dissociation. The apo and the holo forms of the triple mutant R118A-Mi/F238S-Mi/F240S-Mi display a dimer-monomer equilibrium dissociation constant value at least ~260- and 31-fold larger, respectively, than the corresponding ones of AGT-Mi. In the presence of PLP, the apomonomer of the triple mutant undergoes a biphasic process: the fast phase represents the formation of an inactive PLP-bound monomer, while the slow phase depicts the monomer-monomer association that parallels the regain of transaminase activity. The latter events occur with a rate constant of ~0.02 μM-1min-1. In the absence of PLP, the apomonomer is also able to dimerize but with a rate constant value ~2700-fold lower. Thereafter, the possible interference with the dimerization process of AGT-Mi exerted by the mutated residues in the I244T-Mi and F152I-Mi variants associated with Primary Hyperoxaluria type 1 was investigated by molecular dynamics simulations. On the basis of the present and previous studies, a model for the dimerization process of AGT-Mi, I244T-Mi and F152I-Mi, which outlines the structural defects responsible for the complete or partial mistargeting of the pathogenic variants, was proposed and discussed.
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