3 years ago

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

Yasuo Yoshida, Tetsuo Ishida, Yuichiro Kezuka, Takamasa Nonaka
Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from L-cysteine in addition to pyruvate and NH4+ 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. This study leads to elucidation of the H2S-producing mechanism in F. nucleatum.

Publisher URL: http://doi.org/10.1042/BCJ20170838

DOI: 10.1042/BCJ20170838

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