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  • Title: Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.
    Author: Kezuka Y, Ishida T, Yoshida Y, Nonaka T.
    Journal: Biochem J; 2018 Feb 16; 475(4):733-748. PubMed ID: 29343611.
    Abstract:
    Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from l-cysteine in addition to pyruvate and [Formula: see text] 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.
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