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

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.

Keywords: 2‐aminoacrylate; 2‐imino acids; RidA; metabolic damage; protein stability; reactive intermediate deaminase A; x‐ray crystallography.

FIGURE 1

(a) Multiple alignment of RidA protein sequences from Salmo salar‐isoform 1 (A0A1S3KNQ3), S. salar‐isoform 2 (C0H8I4), Capra hircus (P80601), Homo sapiens (P52758), Mus musculus (P52760), Oryctolagus cuniculus (G1U8R2), Arabidopsis thaliana (Q94JQ4), and Escherichia coli (P0AF93). Protein sequences were analyzed using Clustal Omega and ESPript3. Fully conserved residues are highlighted in red; partially conserved residues are in red; blue frames indicate sequence stretches with globally high similarity. Blue stars indicate residues that have been mutated. The N‐terminal signal peptide sequence of A. thaliana was omitted from the alignment. (b,c) Ribbon representation of _ChRidA homotrimeric structure (PDB ID: 1NQ3) (b) and of a _ChRidA monomer with residues mutated in this study shown as spheres (c).

FIGURE 2

Summary of the effects of amino acid substitutions on the substrate specificity of the RidA forms. (a) The activity of RidA variants, as measured from the initial velocity of semicarbazone formation in coupled assays containing different L‐amino acids, LAAO, semicarbazide, and varying amounts of RidA forms (v_RidA), is expressed as percentage of the initial velocity of semicarbazone formation in the absence of RidA (v_o). Symbols and color code are as follows: L‐Leu, red circles; L‐Ala, blue squares; L‐His, magenta stars; L‐Phe, green triangles; L‐Trp, orange inverted triangles; L‐Glu, purple diamonds. Full symbols and empty symbols of different sizes indicate independent experiments. No curves were drawn when the RidA variant had no detectable activity. (b) Catalytic efficiency of each RidA variant (100/K₅₀) with respect to the imino acids derived from six L‐amino acids. The inset panel allows to inspect the low‐activity values exhibited by some of the RidA variants. Only V25W exhibited detectable activity with the imino acid derived from L‐His.

FIGURE 3

(a) Ribbon representation of the superposition of wt and mutant _ChRidA trimers. Color code: Wt _ChRidA, gray; _ChRidA‐R107A, red; _ChRidA‐R107K, orange; _ChRidA‐R107W, yellow; _ChRidA‐K78A, blue; _ChRidA‐E124A, cyan; _ChRidA‐V25W, pink; _ChRidA‐A108D, purple; _ChRidA‐I126Y, green. (b) Ribbon representation of the superposition of wt and variant _ChRidA monomers using the same color code as in (a).

FIGURE 4

(a–c) Representation of active site of _ChRidA‐R107A (red, a), _ChRidA‐R107K (orange, b) and _ChRidA‐R107W (yellow, c) in comparison with wt _ChRidA. Dotted lines represent the interatomic distances between atoms. (d–f) Representation of the inter‐monomeric interface of wt _ChRidA (gray, d), _ChRidA‐K78A (blue, e), and _ChRidA‐E124A (light blue, f). The salt bridge present in wt _ChRidA is represented as dotted line. (g) Comparison of residues orientation of _ChRidA‐V25W (pink) and wt _ChRidA (gray) in the proximity of V25W mutation. (h) Comparison of residues orientation of _ChRidA‐A108D (purple) and wt _ChRidA (gray) in the proximity of A108D mutation. Salt bridges are represented as dotted lines. (i) Comparison of residues orientation and central cavity filling of _ChRidA‐I126Y (green) and wt _ChRidA (gray). In _ChRidA‐I126Y the three monomers are represented with different shades of green.