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

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...

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Bibliographic Details
Published in:Protein science 2024-06, Vol.33 (6), p.e5036-n/a
Main Authors: Rizzi, Giulia, Digiovanni, Stefania, Degani, Genny, Barbiroli, Alberto, Di Pisa, Flavio, Popolo, Laura, Visentin, Cristina, Vanoni, Maria Antonietta, Ricagno, Stefano
Format: Article
Language:English
Subjects:
2‐aminoacrylate
2‐imino acids
Amino acids
Ammonia
Animals
Capra hircus
Catalytic activity
Catalytic Domain
Crystallography
E coli
Enzymatic activity
Enzyme Stability
metabolic damage
Models, Molecular
Mutagenesis
Mutagenesis, Site-Directed
protein stability
Protein structure
Pyruvic acid
Quaternary structure
reactive intermediate deaminase A
RidA
Substrate Specificity
Substrates
Thermal stability
x‐ray crystallography
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Summary: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.
ISSN:0961-8368
1469-896X
DOI:10.1002/pro.5036