Identification of an l-methionine γ-lyase involved in the production of hydrogen sulfide from l-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586

Abstract

Fusobacterium nucleatum produces an abundance of hydrogen sulfide (H(2)S) in the oral cavity that is mediated by several enzymes. The identification and characterization of three distinct enzymes (Fn0625, Fn1055 and Fn1220) in F. nucleatum that catalyse the production of H(2)S from l-cysteine have been reported. In the current study, a novel enzyme involved in the production of H(2)S in F. nucleatum ATCC 25586, whose molecular mass had been estimated to be approximately 130 kDa, was identified by two-dimensional electrophoresis combined with MALDI-TOF MS. The enzyme, Fn1419, has previously been characterized as an l-methionine γ-lyase. SDS-PAGE and gel-filtration chromatography indicated that Fn1419 has a molecular mass of 43 kDa and forms tetramers in solution. Unlike other enzymes associated with H(2)S production in F. nucleatum, the quaternary structure of Fn1419 was not completely disrupted by exposure to SDS. The purified recombinant enzyme exhibited a K(m) of 0.32±0.02 mM and a k(cat) of 0.69±0.01 s(-1). Based on current and published data, the enzymic activity for H(2)S production from l-cysteine in F. nucleatum is ranked as follows: Fn1220>Fn1055>Fn1419>Fn0625. Based on kinetic values and relative mRNA levels of the respective genes, as determined by real-time quantitative PCR, the amount of H(2)S produced by Fn1419 was estimated to be 1.9 % of the total H(2)S produced from l-cysteine in F. nucleatum ATCC 25586. In comparison, Fn1220 appeared to contribute significantly to H(2)S production (87.6 %).