Cysteine Dealkylation in Bacillus subtilis by a Novel Flavin-Dependent Monooxygenase

Abstract

In this paper, we describe the biochemical reconstitution of a cysteine salvage pathway and the biochemical characterization of each of the five enzymes involved. The salvage begins with amine acetylation of S-alkylcysteine, followed by thioether oxidation. The C–S bond of the resulting sulfoxide is cleaved using a new flavoenzyme catalytic motif to give N-acetylcysteine sulfenic acid. This is then reduced to the thiol and deacetylated to complete the salvage pathway. We propose that this pathway is important in the catabolism of alkylated cysteine generated by proteolysis of alkylated glutathione formed in the detoxification of a wide range of electrophiles.

Alkylcysteine Sulfoxide C–S Monooxygenase Uses a Flavin-Dependent Pummerer Rearrangement

“…Previous mechanistic studies on the photooxidation of thioethers suggested a third possible mechanism. 29 These studies demonstrated that thioether photooxidation proceeds via a persulfoxide intermediate 28 which then undergoes a Pummerer rearrangement, via intermediate 29, to give α- Mechanism III is consistent with our previous observation that the aldehyde oxygen is derived from molecular oxygen 14 and predicts that the oxygen of the sulfenic acid product 4 is also derived from molecular oxygen and not from the sulfoxide substrate 13. Analysis of the source of the sulfenic acid oxygen is complex because sulfenic acids are highly reactive compounds with both nucleophilic and electrophilic reactivity and undergo rapid dimerization and reduction reactions.…”

“…While it has been reported that such S-conjugates undergo intracellular or extracellular hydrolysis that eventually leads to the formation of cysteine S-conjugates, ,− the fate of these cysteine derivatives was not known. Our previous report suggests the role of soil bacteria in the catabolism of such cysteine S-conjugates, by salvaging the cysteine. It had been shown that the pathway utilizes two flavoenzymes (CmoO and CmoJ) to activate and cleave the C–S bond.…”

“…CmoJ then catalyzes the cleavage of this sulfoxide to give sulfenic acid 4 . Reduction of 4 followed by deacetylation completes the salvage pathway . The C–S bond cleavage reaction shown in the conversion of 3 to 4 is an unprecedented flavoenzyme-mediated C–S bond cleavage.…”

“…12,13 CmoJ, which is a sequence homologue of YxeK and DszA, is involved in salvaging cysteine from S-alkylated cysteines. 14,15 S-Alkylated cysteine is formed in the dealkylation of DNA, 16 and in the salvage of cysteine from alkylated glutathione and related low molecular weight thiols involved in the detoxification of environmental alkylating agents. 17 The recently discovered cysteine salvage pathway 15 shown in Figure 1 14 is widely distributed in soil bacteria.…”

Identification of a S-(2-succino)cysteine breakdown pathway that uses a novel S-(2-succino) lyase

“…YxeK and 2SL represent two variants of the pathway that use completely different enzymatic activities to perform the breakdown step. This general strategy—acetylation, breakdown, and deacetylation—is used for the breakdown of other S -alkyl adducts of cysteine such as S -methylcysteine ( 41 , 42 ).…”