Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum

Hydrogen sulfide (H₂S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H₂S from l-cysteine in addition to pyruvate and ⁠. However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the production of H₂S and l-serine from l-cysteine and H₂O, an unusual cysteine (hydroxyl) lyase reaction (β-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp²³² and Ser⁷⁴, and the substrate α-carboxylate interacts with Thr⁷³ and Gln¹⁴⁷. Asp²³² is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of β-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp²³² is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp²³² is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser⁷⁴, Thr⁷³, and Gln¹⁴⁷) exhibited reduced β-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H₂S-producing mechanism in F. nucleatum.