title: Effects of interface mutations on the dimerization of alanine glyoxylate aminotransferase and implications in the mistargeting of the pathogenic variants F152I and I244T.
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.
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Verona, Italy.
authors.forename: Mirco
authors.initials: M
authors.lastname: Dindo
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Verona, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Montioli
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Busato
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Giorgetti
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Verona, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Verona, Italy. Electronic address: carla.borrivoltattorni@univr.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Borri Voltattorni
mesh_terms: D000465:Algorithms; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011732:Pyridoxal Phosphate; D000637:Transaminases; C019635:Alanine-glyoxylate transaminase
keywords: Human alanine glyoxylate aminotransferase; Interface hot spots; Molecular dynamics; Primary hyperoxaluria type I; Protein dimerization; Protein mistargeting