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.

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]

Site-directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus

G. Rizzi
Primo
;
S. Digiovanni;G. Degani;A. Barbiroli;L. Popolo;C. Visentin
;
M.A. Vanoni
Penultimo
;
S. Ricagno
Ultimo
2024

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.
2‐aminoacrylate; 2‐imino acids; RidA; metabolic damage; protein stability; reactive intermediate deaminase A; x‐ray crystallography
Settore BIO/10 - Biochimica
   Protein misfolding in AL amyloidosis: from fibrillar deposits to soluble toxicity
   MolAL
   MINISTERO DELL'ISTRUZIONE E DEL MERITO
   20207XLJB2_001
giu-2024
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/1054631
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