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

• Published in: Archives of biochemistry and biophysics Vol. 746; p. 109727
• Main Authors: Darbyshire, Amanda L., Wolthers, Kirsten R.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 15.09.2023
• Subjects: Fusobacterium nucleatum; H2S; Pyridoxal 5′-phosphate; Serine synthase; Serine synthase; H2S; Pyridoxal 5′-phosphate; Fusobacterium nucleatum
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2023.109727

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5′-phosphate-dependent enzyme that catalyzes the reversible β-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 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 Asp232 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 (Trp99, Phe125, Phe148 and Phe234) 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 ⍺-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 β-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3–4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-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. [Display omitted] •Serine synthase catalyzes the reversible β-replacement of l-cysteine and l-serine.•Enzyme forms aminoacylate upon reaction with l-serine.•Hydrophobic residues increase the basicity of D232 for activation of a water.•Mutation of F125 and F148 enables other nucleophiles to react with aminoacrylate.•Substitutions increase potential for noncanonical amino acid biosynthesis.