Expanding the β-substitution reactions of serine synthase through mutagenesis of aromatic active site residues

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible beta-replacement of L-cysteine and L-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp(232) that serves as a general base in the activation of a water molecule for nucleophilic attack of the.-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp(232) are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the.-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp(99), Phe(125), Phe(148) and Phe(234)) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the alpha-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the.-aminoacrylate intermediate upon reaction with either L-cysteine or L-serine; however, the rate constant associated with the elimination of the beta-hydroxyl group from L-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe(125) and Phe(148), situated similar to 3-4 angstrom from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the alpha-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for L-cysteine, decrease the binding of the product, L-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.

Automated summary

In Fusobacterium nucleatum, the clustering of aromatic residues is critical for the catalytic activity of serine synthase, an enzyme involved in the biosynthesis of noncanonical amino acids. Substituting these aromatic residues affects the reaction rate and the scope of nucleophilic substrates.