Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586

Abstract

Fusobacterium nucleatumproduces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis ofF. nucleatumH2S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts fromF. nucleatumATCC 25586 contained three major H2S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H2S froml-cysteine (∼30 %) than Fn1220. The Fn0625 protein degraded a variety of substrates containingβC–S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia froml-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation ofl-cysteine with Fn1220 produced H2S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans ofF. nucleatumATCC 25586. In contrast, most of the sulfur-containing substrates tested, exceptl-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that thefn1220gene showed several-fold higher expression thanfn0625and housekeeping genes in exponential-phase cultures ofF. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H2S in distinct manners: Fn0625 carries outβ-elimination ofl-cysteine to produce H2S, pyruvate and ammonia, whereas Fn1220 catalyses theβ-replacement ofl-cysteine to produce H2S and lanthionine, the latter of which may be used for peptidoglycan formation inF. nucleatum.