Site-directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus
Articolo
Data di Pubblicazione:
2024
Citazione:
Site-directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus / G. Rizzi, S. Digiovanni, G. Degani, A. Barbiroli, F. Di Pisa, L. Popolo, C. Visentin, M.A. Vanoni, S. Ricagno. - In: PROTEIN SCIENCE. - ISSN 1469-896X. - 33:6(2024 Jun), pp. e5036.1-e5036.12. [10.1002/pro.5036]
Abstract:
Reactive intermediate deaminase A (RidA) is a highly conserved enzyme that catalyzes the hydrolysis of 2-imino acids to the corresponding 2-keto acids and ammonia. RidA thus prevents the accumulation of such potentially harmful compounds in the cell, as exemplified by its role in the degradation of 2-aminoacrylate, formed during the metabolism of cysteine and serine, catalyzing the conversion of its stable 2-iminopyruvate tautomer into pyruvate. Capra hircus (goat) RidA (ChRidA) was the first mammalian RidA to be isolated and described. It has the typical homotrimeric fold of the Rid superfamily, characterized by remarkably high thermal stability, with three active sites located at the interface between adjacent subunits. ChRidA exhibits a broad substrate specificity with a preference for 2-iminopyruvate and other 2-imino acids derived from amino acids with non-polar non-bulky side chains. Here we report a biophysical and biochemical characterization of eight ChRidA variants obtained by site-directed mutagenesis to gain insight into the role of specific residues in protein stability and catalytic activity. Each mutant was produced in Escherichia coli cells, purified and characterized in terms of quaternary structure, thermal stability and substrate specificity. The results are rationalized in the context of the high-resolution structures obtained by x-ray crystallography.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
2‐aminoacrylate; 2‐imino acids; RidA; metabolic damage; protein stability; reactive intermediate deaminase A; x‐ray crystallography
Elenco autori:
G. Rizzi, S. Digiovanni, G. Degani, A. Barbiroli, F. Di Pisa, L. Popolo, C. Visentin, M.A. Vanoni, S. Ricagno
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