Please use this identifier to cite or link to this item: http://hdl.handle.net/20.500.12857/116341
Title: Effects of interface mutations on the dimerization of alanine glyoxylate aminotransferase and implications in the mistargeting of the pathogenic variants F152I and I244T
Authors: Dindo, Mirco 
Montioli, Riccardo 
Busato, Mirko 
Giorgetti, Alejandro 
Cellini, Barbara 
Borri Voltattorni, Carla 
Keywords: Human alanine glyoxylate aminotransferase;Interface hot spots;Molecular dynamics;Primary hyperoxaluria type I;Protein dimerization;Protein mistargeting
Keywords Plus: MITOCHONDRIAL TARGETING SEQUENCE;PRIMARY HYPEROXALURIA TYPE-1;MOLECULAR-DYNAMICS;DIMERIC STRUCTURE;AGGREGATION;STABILITY;PHENOTYPE;DIAGNOSIS;GENOTYPE;IMPACT
Mesh headings: Mutation;Protein Multimerization;Transaminases
Secondary Mesh headings: Algorithms;Amino Acid Substitution;Circular Dichroism;Humans;Hydrophobic and Hydrophilic Interactions;Kinetics;Molecular Dynamics Simulation;Protein Binding;Protein Domains;Pyridoxal Phosphate;Spectrometry, Fluorescence
Issue Date: Dec-2016
Publisher: ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
Journal: Biochimie 
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.
URI: http://hdl.handle.net/20.500.12857/116341
ISSN: 03009084
DOI: 10.1016/j.biochi.2016.10.001
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