Unusual flavoenzyme catalysis in marine bacteria

Abstract

Ever since the discovery of the flavin cofactor more than 80 years ago, flavin-dependent enzymes have emerged as ubiquitous and versatile redox catalysts in primary metabolism. Yet, the recent advances in the discovery and characterization of secondary metabolic pathways exposed new roles for flavin-mediated catalysis in the generation of structurally complex natural products. Here, we review a selection of key biosynthetic flavoenzymes from marine bacterial secondary metabolism and illustrate how their functional and mechanistic investigation expanded our view of the cofactor's chemical repertoire and led to the discovery of a previously unknown flavin redox state.

Figure 1. Overview of flavin redox states and catalysis

(R = ribityl-ADP (FAD) or phosphoribityl (FMN)). Oxidases and dehydrogenases (DHase) oxidize organic substrates (S1) and utilize O₂ or alternative substrates (S2) for the reoxidation of Fl_red to the catalytically active Fl_ox, respectively. Monooxygenases employ a flavin-C4a-(hydro)peroxide or a flavin-N5-oxide for the oxygenation of organic substrates (S1). The radical formation of these oxygenating species proceeds via reduction of Fl_ox to Fl_red by NAD(P)H or the substrate, which may enable the single electron reduction of O₂ and subsequent radical coupling of the formed superoxide and the flavin SQ at the spin density sites C4a or N5. Note that the tentative pathway for Fl_N5[O] formation requires further investigation.

Figure 2. Flavin-dependent halogenation chemistry

(A) Reaction mechanism for flavin-dependent halogenases. Flavin-dependent halogenases are O₂-dependent enzymes, and the first half of the reaction is identical to flavin-dependent oxygenases, in which the transient Fl_C4a[OOH] transient species is generated. This decomposes to a postulated Fl_C4a[OX] transition state with the concomitant release of a water molecule, whereupon the halonium is transferred to an appropriately positioned catalytic lysine side chain. The lysine side chain halo-amine is postulated to be the active halogenating agent. Note that in canonical flavin-dependent halogenases, Fl_red is provided by a partner flavin-reductase enzyme. (B) Active site representation of the canonical flavin-dependent halogenase PrnA (37) in complex with cofactor FAD (in stick-ball representation with carbon atoms colored yellow), a chloride ion (in sphere representation colored orange), and the product 6-chloro-tryptophan (in stick ball representation with carbon atoms colored blue). Also shown are the haloamine bearing lysine side chain, the catalytic base glutamate side chain that abstracts the proton subsequent to chloronium addition to the substrate, and the water molecules in close proximity to the chloride ion. The C4 and N5 positions of the flavin isoalloxazine ring are marked. (C) Reactions catalyzed by Bmp2 and Mpy16. (D) Two-step reaction for Bmp5 leading to the formation of 2,4-dibromophenol from p-hydroxybenzoic acid. 2,4-dibromophenol further undergoes oxidative coupling to generate bioaccummulative and persistent pollutant natural products such as the polybrominated diphenyl ethers 2OH-BDE68 and biphenyl-2,2′-diOH-BB80.

Figure 3. Selected flavin-catalyzed coupling reactions

(A) Proposed pyrrolizine ring formation by Clz9. Prechlorizidine A is dehydrogenated by Clz9-Fl_ox, followed by the regio- and stereoselective pyrrolizine-forming cyclization reaction through pyrrole coupling that affords chlorizidine A. The C8α and C6 of the flavin cofactor are likely covalently linked to His99 and Cys157 of Clz9, respectively. Oxidation of Fl_red by O₂ regenerates Fl_ox (not shown). (B) Tentative mechanism for the XiaH-catalyzed radical xiamycin coupling and oxiamycin formation. XiaH is proposed to catalyze the single-electron oxidation of the indoloterpenoid xiamycin, which then undergoes various radical dimerization reactions to bixiamycins or reacts with superoxide and possibly NAD(P)H to oxiamycin. The asterisks indicate a mixture of the respective atropoisomers with stable P- and M-configurations. (C) Proposed mechanism for C-N bond formation by the Mpy10-Mpy11 enzyme pair. Cryptic halogenation at either the C3 or the N1 positions of the monodeoxypyoluteorin monomer leads to the formation of a trihalo intermediate that undergoes nucleophilic halide displacement to yield the marinopyrrole dimer. Note that a radical mechanism is plausible too.

Figure 4. Proposed EncM-Fl_N5[O]-catalyzed poly(β)-ketone dual oxidation

The C8α of the flavin cofactor is covalently attached to the His78 of EncM. Note that the EncM-mediated Favorskii rearrangement and subsequent reactions required for enterocin-formation are not shown. The steps indicated by the roman numerals are discussed in the main text. For details of the Fl_N5[O]-regeneration pathway see Figure 1.