Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis.

Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.

From Tryptophan to Toxin: Nature's Convergent Biosynthetic Strategy to Aetokthonotoxin.

Aetokthonotoxin (AETX) is a cyanobacterial neurotoxin that causes vacuolar myelinopathy, a neurological disease that is particularly deadly to bald eagles in the United States. The recently characterized AETX is structurally unique among cyanotoxins and is composed of a pentabrominated biindole nitrile. Herein we report the discovery of an efficient, five-enzyme biosynthetic pathway that the freshwater cyanobacterium Aetokthonos hydrillicola uses to convert two molecules of tryptophan to AETX. We demonstrate that the biosynthetic pathway follows a convergent route in which two functionalized indole monomers are assembled and then reunited by biaryl coupling catalyzed by the cytochrome P450 AetB. Our results revealed enzymes with novel biochemical functions, including the single-component flavin-dependent tryptophan halogenase AetF and the iron-dependent nitrile synthase AetD.

Cryptic halogenation reactions in natural product biosynthesis.

Covering: Up to December 2020Enzymatic halogenation reactions are essential for the production of thousands of halogenated natural products. However, in recent years, scientists discovered several halogenases that transiently incorporate halogen atoms in intermediate biosynthetic molecules to activate them for further chemical reactions such as cyclopropanation, terminal alkyne formation, C-/O-alkylation, biaryl coupling, and C-C rearrangements. In each case, the halogen atom is lost in the course of biosynthesis to the final product and is hence termed "cryptic". In this review, we provide an overview of our current knowledge of cryptic halogenation reactions in natural product biosynthesis.

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation.

HcbA1 is a unique flavoenzyme that catalyzes the first step in the bacterial hexachlorobenzene catabolic pathway. Here we report in vitro reconstitution of the HcbA1-catalyzed reaction. Detailed mechanistic studies provide evidence for nucleophilic aromatic substitution and flavin-N5-oxide formation.

RutA-Catalyzed Oxidative Cleavage of the Uracil Amide Involves Formation of a Flavin-N5-oxide.

RutA is a novel flavoenzyme on the uracil catabolic pathway that catalyzes uracil ring opening by a unique amide oxidation reaction. Here we provide evidence that this reaction also involves the formation of a flavin-N5-oxide.

Flavin-N5-oxide: A new, catalytic motif in flavoenzymology.

Flavin-N5-oxide is a recently discovered intermediate used by EncM (1,3-diketone oxidation), DszA (sulfone monooxygenase) and RutA (amide monooxygenase). This review describes the mechanism of these enzymes and proposes criteria for the identification of additional Flavin-N5-oxide dependent enzymes.

Dibenzothiophene Catabolism Proceeds via a Flavin-N5-oxide Intermediate.

The dibenzothiophene catabolic pathway converts dibenzothiophene to 2-hydroxybiphenyl and sulfite. The third step of the pathway, involving the conversion of dibenzothiophene sulfone to 2-(2-hydroxyphenyl)-benzenesulfinic acid, is catalyzed by a unique flavoenzyme DszA. Mechanistic studies on this reaction suggest that the C2 hydroperoxide of dibenzothiophene sulfone reacts with flavin to form a flavin-N5-oxide. The intermediacy of the flavin-N5-oxide was confirmed by LC-MS analysis, a co-elution experiment with chemically synthesized FMN-N5-oxide and (18)O2 labeling studies.

Oxidative rearrangement of tryptophan to indole nitrile by a single diiron enzyme.

Nitriles are uncommon in nature and are typically constructed from oximes via the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third strategy of nitrile biosynthesis featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the 'eagle-killing' neurotoxin, aetokthonotoxin, AetD converts the alanyl side chain of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical, and biophysical techniques, we characterized AetD as a non-heme diiron enzyme that belongs to the emerging Heme Oxygenase-like Diiron Oxidase and Oxygenase (HDO) superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation that we propose proceeds via an aziridine intermediate. Our work presents a new paradigm for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other HDO enzymes.

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis.

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis with and without attachment in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2. Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium's ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides. We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for coral restoration.

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis.

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2 . Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium's ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides . We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for restoring degraded coral reefs.

Flavin-N5-oxide intermediates in dibenzothiophene, uracil, and hexachlorobenzene catabolism.

Flavin-N5-oxide is a new intermediate in flavoenzymology. Here we describe the identification of DszA (dibenzothiophene catabolism), RutA (uracil catabolism) and HcbA1 (hexachlorobenzene catabolism) as flavin-N5-oxide-utilizing enzymes. Mechanistic analysis of these reactions suggests a model for the identification of other examples of this catalytic motif.