Characterization of a Radical SAM Oxygenase for the Ether Crosslinking in Darobactin Biosynthesis

Abstract

Darobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C–C crosslinking. However, the specific mechanism of the crosslink formation, especially the ether crosslink, remains elusive. Here, using in vitro enzyme assays, we demonstrate that both crosslinks are formed by the DarE radical S-adenosylmethionine (SAM) enzyme in an O2-dependent manner. The relevance of the observed activity to darobactin A biosynthesis was demonstrated by proteolytic transformation of the DarE product into darobactin A. Furthermore, DarE assays in the presence of 18O2 or [18O]­water demonstrated that the oxygen of the ether crosslink originates from O2 and not from water. These results demonstrate that DarE is a radical SAM enzyme that uses oxygen as a co-substrate in its physiologically relevant function. Since radical SAM enzymes are generally considered to function under anaerobic environments, the discovery of a radical SAM oxygenase represents a significant change in the paradigm and suggests that these radical SAM enzymes function in aerobic cells. Also, the study revealed that DarE catalyzes the formation of three distinct modifications on DarA; ether and C–C crosslinks and α,β-desaturation. Based on these observations, possible mechanisms of the DarE-catalyzed reactions are discussed.

Substrate-Controlled Catalysis in the Ether Cross-Link-Forming Radical SAM Enzymes

“…To obtain sufficient amount of 1 for further structural characterization, we tried on different commercially available proteases and found that proteinase K can efficiently release 1 from modified PasA in vitro (Figure S11), in a way similar to the recent work on darobactin . We scaled up the expression, digestion, and purification process, which afforded 1.5 mg of 1 for NMR characterization.…”

“…(A) Maximum likelihood phylogenetic tree of the daropeptide precursors. The daropeptide precursors investigated in previous studies (i.e., pk -DarA, , pl -DarA, and yi -DarA) and in this study (i.e., PasA and RscA) are marked by white circle and yellow circle, respectively. Key bootstrap values for tree construction are indicated along the branch.…”

“…The assays were also performed with or without additional supplement of O 2 (Figure S32), as suggested by Yokoyama and co-workers. 10 The assays are quenched via heat-inactivation and coupled with proteinase K digestion (Figure S33). The results showed that PasB completely modifies PasA to the +14 Da product in a full reaction in which FldAR/NADPH is used as a reductant, and O 2 is supplemented in the reaction (Figures 5A,B and S32 and S33).…”

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB

“…OxsB then catalyzes dehydrogenation of the C2′-CH 3 methyl to yield the methylidene 10-P rather than a second round of methylation as observed in other radical-mediated methyltransferases. − Although examples of radical SAM catalyzed dehydrogenation of alcohols, thiols, and amines are well-known, − biological C-CH 3 dehydrogenase activity has not been previously reported for a radical SAM enzyme. Nevertheless, NosL from nosiheptide biosynthesis catalyzes C–C desaturation of a substrate structural analogue, and DarE from the darobactin maturation pathway has been identified to catalyze off-pathway dehydrogenation of the C α –C β bond of a tryptophan residue in its peptide substrate. , Once formed, 10-P could then serve as an acceptor for OxsB catalyzed addition of the 5′-deoxyadenosyl radical ( 2 ). This chemistry is effectively equivalent to that of MqnE and QueE requiring ejection of one electron from the reduced radical adduct such that the overall addition (i.e., 10-P + SAM → 11-P + Met) is redox neutral .…”

“…Nevertheless, NosL from nosiheptide biosynthesis catalyzes C−C desaturation of a substrate structural analogue, 39 and DarE from the darobactin maturation pathway has been identified to catalyze off-pathway dehydrogenation of the C α −C β bond of a tryptophan residue in its peptide substrate. 40,41 Once formed, 10-P could then serve as an acceptor for OxsB catalyzed addition of the 5′deoxyadenosyl radical (2). This chemistry is effectively equivalent to that of MqnE 42 and QueE 43 requiring ejection of one electron from the reduced radical adduct such that the overall addition (i.e., 10-P + SAM → 11-P + Met) is redox neutral.…”