Biosynthesis of the bacterial antibiotic 3,7-dihydroxytropolone through enzymatic salvaging of catabolic shunt products.

The non-benzenoid aromatic tropone ring is a structural motif of numerous microbial and plant natural products with potent bioactivities. In bacteria, tropone biosynthesis involves early steps of the widespread CoA-dependent phenylacetic acid (paa) catabolon, from which a shunt product is sequestered and surprisingly further utilized as a universal precursor for structurally and functionally diverse tropone derivatives such as tropodithietic acid or (hydroxy)tropolones. Here, we elucidate the biosynthesis of the antibiotic 3,7-dihydroxytropolone in Actinobacteria by in vitro pathway reconstitution using paa catabolic enzymes as well as dedicated downstream tailoring enzymes, including a thioesterase (TrlF) and two flavoprotein monooxygenases (TrlCD and TrlE). We furthermore mechanistically and structurally characterize the multifunctional key enzyme TrlE, which mediates an unanticipated ipso-substitution involving a hydroxylation and subsequent decarboxylation of the CoA-freed side chain, followed by ring oxidation to afford tropolone. This study showcases a remarkably efficient strategy for 3,7-dihydroxytropolone biosynthesis and illuminates the functions of the involved biosynthetic enzymes.

Bacterial Dehydrogenases Facilitate Oxidative Inactivation and Bioremediation of Chloramphenicol.

Antimicrobial resistance represents a major threat to human health and knowledge of the underlying mechanisms is therefore vital. Here, we report the discovery and characterization of oxidoreductases that inactivate the broad-spectrum antibiotic chloramphenicol via dual oxidation of the C3-hydroxyl group. Accordingly, chloramphenicol oxidation either depends on standalone glucose-methanol-choline (GMC)-type flavoenzymes, or on additional aldehyde dehydrogenases that boost overall turnover. These enzymes also enable the inactivation of the chloramphenicol analogues thiamphenicol and azidamfenicol, but not of the C3-fluorinated florfenicol. Notably, distinct isofunctional enzymes can be found in Gram-positive (e. g., Streptomyces sp.) and Gram-negative (e. g., Sphingobium sp.) bacteria, which presumably evolved their selectivity for chloramphenicol independently based on phylogenetic analyses. Mechanistic and structural studies provide further insights into the catalytic mechanisms of these biotechnologically interesting enzymes, which, in sum, are both a curse and a blessing by contributing to the spread of antibiotic resistance as well as to the bioremediation of chloramphenicol.

An acetyltransferase controls the metabolic flux in rubromycin polyketide biosynthesis by direct modulation of redox tailoring enzymes.

The often complex control of bacterial natural product biosynthesis typically involves global and pathway-specific transcriptional regulators of gene expression, which often limits the yield of bioactive compounds under laboratory conditions. However, little is known about regulation mechanisms on the enzymatic level. Here, we report a novel regulatory principle for natural products involving a dedicated acetyltransferase, which modifies a redox-tailoring enzyme and thereby enables pathway furcation and alternating pharmacophore assembly in rubromycin polyketide biosynthesis. The rubromycins such as griseorhodin (grh) A are complex bioactive aromatic polyketides from Actinobacteria with a hallmark bisbenzannulated [5,6]-spiroketal pharmacophore that is mainly installed by two flavoprotein monooxygenases. First, GrhO5 converts the advanced precursor collinone into the [6,6]-spiroketal containing dihydrolenticulone, before GrhO6 effectuates a ring contraction to afford the [5,6]-spiroketal. Our results show that pharmacophore assembly in addition involves the acetyl-CoA-dependent acetyltransferase GrhJ that activates GrhO6 to allow the rapid generation and release of its labile product, which is subsequently sequestered by ketoreductase GrhO10 and converted into a stable intermediate. Consequently, the biosynthesis is directed to the generation of canonical rubromycins, while the alternative spontaneous [5,6]-spiroketal hydrolysis to a ring-opened pathway product is thwarted. Presumably, this allows the bacteria to rapidly adjust the biosynthesis of functionally distinct secondary metabolites depending on nutrient and precursor (i.e. acetyl-CoA) availability. Our study thus illustrates how natural product biosynthesis can be enzymatically regulated and provides new perspectives for the improvement of in vitro enzyme activities and natural product titers via biotechnological approaches.

Three Rings to Rule Them All: How Versatile Flavoenzymes Orchestrate the Structural Diversification of Natural Products.

The structural diversification of natural products is instrumental to their versatile bioactivities. In this context, redox tailoring enzymes are commonly involved in the modification and functionalization of advanced pathway intermediates en route to the mature natural products. In recent years, flavoprotein monooxygenases have been shown to mediate numerous redox tailoring reactions that include not only (aromatic) hydroxylation, Baeyer-Villiger oxidation, or epoxidation reactions but also oxygenations that are coupled to extensive remodeling of the carbon backbone, which are often central to the installment of the respective pharmacophores. In this Perspective, we will highlight recent developments and discoveries in the field of flavoenzyme catalysis in bacterial natural product biosynthesis and illustrate how the flavin cofactor can be fine-tuned to enable chemo-, regio-, and stereospecific oxygenations via distinct flavin-C4a-peroxide and flavin-N5-(per)oxide species. Open questions remain, e.g., regarding the breadth of chemical reactions enabled particularly by the newly discovered flavin-N5-oxygen adducts and the role of the protein environment in steering such cascade-like reactions. Outstanding cases involving different flavin oxygenating species will be exemplified by the tailoring of bacterial aromatic polyketides, including enterocin, rubromycins, rishirilides, mithramycin, anthracyclins, chartreusin, jadomycin, and xantholipin. In addition, the biosynthesis of tropone natural products, including tropolone and tropodithietic acid, will be presented, which features a recently described prototypical flavoprotein dioxygenase that may combine flavin-N5-peroxide and flavin-N5-oxide chemistry. Finally, structural and mechanistic features of selected enzymes will be discussed as well as hurdles for their application in the formation of natural product derivatives via bioengineering.

Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis.

The medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]-spiroketal pharmacophore that was recently shown to be assembled by flavin-dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]-spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon-carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals.

A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation.

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).

The scope of flavin-dependent reactions and processes in the model plant Arabidopsis thaliana.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are utilized as coenzymes in many biochemical reduction-oxidation reactions owing to the ability of the tricyclic isoalloxazine ring system to employ the oxidized, radical and reduced state. We have analyzed the genome of Arabidopsis thaliana to establish an inventory of genes encoding flavin-dependent enzymes (flavoenzymes) as a basis to explore the range of flavin-dependent biochemical reactions that occur in this model plant. Expectedly, flavoenzymes catalyze many pivotal reactions in primary catabolism, which are connected to the degradation of basic metabolites, such as fatty and amino acids as well as carbohydrates and purines. On the other hand, flavoenzymes play diverse roles in anabolic reactions most notably the biosynthesis of amino acids as well as the biosynthesis of pyrimidines and sterols. Importantly, the role of flavoenzymes goes much beyond these basic reactions and extends into pathways that are equally crucial for plant life, for example the production of natural products. In this context, we outline the participation of flavoenzymes in the biosynthesis and maintenance of cofactors, coenzymes and accessory plant pigments (e. g. carotenoids) as well as phytohormones. Moreover, several multigene families have emerged as important components of plant immunity, for example the family of berberine bridge enzyme-like enzymes, flavin-dependent monooxygenases and NADPH oxidases. Furthermore, the versatility of flavoenzymes is highlighted by their role in reactions leading to tRNA-modifications, chromatin regulation and cellular redox homeostasis. The favorable photochemical properties of the flavin chromophore are exploited by photoreceptors to govern crucial processes of plant adaptation and development. Finally, a sequence- and structure-based approach was undertaken to gain insight into the catalytic role of uncharacterized flavoenzymes indicating their involvement in unknown biochemical reactions and pathways in A. thaliana.

The devil is in the details: The chemical basis and mechanistic versatility of flavoprotein monooxygenases.

The ubiquitous flavoenzymes commonly catalyze redox chemistry such as the monooxygenation of organic substrates and are both widely utilized in nature (e.g., in primary and secondary metabolism) and of significant industrial interest. In this work, we highlight the structural and mechanistic characteristics of the distinct types of flavoprotein monooxygenases (FPMOs). We thereby illustrate the chemical basis of FPMO catalysis, which enables reactions such as (aromatic) hydroxylation, epoxidation, (de)halogenation, heteroatom oxygenation, Baeyer-Villiger oxidation, α-hydroxylation of ketones, or non-oxidative carbon-hetero bond cleavage. This seemingly unmatched versatility in oxygenation chemistry results from extensive fine-tuning and regiospecific functionalization of the flavin cofactor that is tightly controlled by the surrounding protein matrix. Accordingly, FPMOs steer the formation of covalent flavin-oxygen adducts for oxygen transfer in the form of the classical flavin-C4a-(hydro)peroxide or the recently discovered N5-functionalized flavins (i.e. the flavin-N5-oxide and the flavin-N5-peroxide), while in rare cases covalent oxygen adduct formation may be foregone entirely. Finally, we speculate about hitherto undiscovered flavin-mediated oxygenation reactions and compare FPMOs to cytochrome P450 monooxygenases, before addressing open questions and challenges for the future investigation of FPMOs.

New inhibitors of chorismate synthase present antifungal activity against Paracoccidioides brasiliensis.

Aim: A structural model of chorismate synthase (CS) from the pathogenic fungus Candida albicans was used for virtual screening simulations. Methods: Docking, molecular dynamics, cell growth inhibition and protein binding assays were used for search and validation. Results: Two molecules termed CS8 and CaCS02 were identified. Further studies of the minimal inhibitory concentration demonstrated fungicidal activity against Paracoccidioides brasiliensis with a minimal inhibitory concentration and minimal fungicidal concentration of 512 and 32 µg·ml-1 for CS8 and CaCS02, respectively. In addition, CaCS02 showed a strong synergistic effect in combination with amphotericin B without cytotoxic effects. In vitro studies using recombinant CS from P. brasiliensis showed IC50 of 29 µM for CaCS02 supporting our interpretation that inhibition of CS causes the observed fungicidal activity.

Biochemical characterization of human D-2-hydroxyglutarate dehydrogenase and two disease related variants reveals the molecular cause of D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.

Closing the gap: yeast electron-transferring flavoprotein links the oxidation of d-lactate and d-α-hydroxyglutarate to energy production via the respiratory chain.

Electron-transferring flavoproteins (ETFs) have been found in all kingdoms of life, mostly assisting in shuttling electrons to the respiratory chain for ATP production. While the human (h) ETF has been studied in great detail, very little is known about the biochemical properties of the homologous protein in the model organism Saccharomyces cerevisiae (yETF). In view of the absence of client dehydrogenases, for example, the acyl-CoA dehydrogenases involved in the ß-oxidation of fatty acids, d-lactate dehydrogenase 2 (Dld2) appeared to be the only relevant enzyme that is serviced by yETF for electron transfer to the mitochondrial electron transport chain. However, this hypothesis was never tested experimentally. Here, we report the biochemical properties of yETF and Dld2 as well as the electron transfer reaction between the two proteins. Our study revealed that Dld2 oxidizes d-α-hydroxyglutarate more efficiently than d-lactate exhibiting kcatapp /KMapp values of 1200 ± 300 m-1 ·s-1 and 11 ± 2 m-1 ·s-1 , respectively. As expected, substrate-reduced Dld2 very slowly reacted with oxygen or the artificial electron acceptor 2,6-dichlorophenol indophenol. However, photoreduced Dld2 was rapidly reoxidized by oxygen, suggesting that the reaction products, that is, α-ketoglutarate and pyruvate, 'lock' the reduced enzyme in an unreactive state. Interestingly, however, we could demonstrate that substrate-reduced Dld2 rapidly transfers electrons to yETF. Therefore, we conclude that the formation of a product-reduced Dld2 complex suppresses electron transfer to dioxygen but favors the rapid reduction in yETF, thus preventing the loss of electrons and the generation of reactive oxygen species.

Promising New Antifungal Treatment Targeting Chorismate Synthase from Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides, is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis (PbCS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 µM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC50) of 47 ± 5 µM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment.

Asymmetric Reductive Carbocyclization Using Engineered Ene Reductases.

Ene reductases from the Old Yellow Enzyme (OYE) family reduce the C=C double bond in α,ß-unsaturated compounds bearing an electron-withdrawing group, for example, a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we show that members of this enzyme family can also catalyze the formation of C-C bonds. α,ß-Unsaturated aldehydes and ketones containing an additional electrophilic group undergo reductive cyclization. Mechanistically, the two-electron-reduced enzyme cofactor FMN delivers a hydride to generate an enolate intermediate, which reacts with the internal electrophile. Single-site replacement of a crucial Tyr residue with a non-protic Phe or Trp favored the cyclization over the natural reduction reaction. The new transformation enabled the enantioselective synthesis of chiral cyclopropanes in up to >99 % ee.

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase.

The berberine bridge enzyme from the California poppy Eschscholzia californica (EcBBE) catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, that is, the formation of the berberine bridge in the biosynthesis of benzylisoquinoline alkaloids. Interestingly, a large number of BBE-like genes have been identified in plants that lack alkaloid biosynthesis. This finding raised the question of the primordial role of BBE in the plant kingdom, which prompted us to investigate the closest relative of EcBBE in Physcomitrella patens (PpBBE1), the most basal plant harboring a BBE-like gene. Here, we report the biochemical, structural, and in vivo characterization of PpBBE1. Our studies revealed that PpBBE1 is structurally and biochemically very similar to EcBBE. In contrast to EcBBE, we found that PpBBE1 catalyzes the oxidation of the disaccharide cellobiose to the corresponding lactone, that is, PpBBE1 is a cellobiose oxidase. The enzymatic reaction mechanism was characterized by a structure-guided mutagenesis approach that enabled us to assign a catalytic role to amino acid residues in the active site of PpBBE1. In vivo experiments revealed the highest level of PpBBE1 expression in chloronema, the earliest stage of the plant's life cycle, where carbon metabolism is strongly upregulated. It was also shown that the enzyme is secreted to the extracellular space, where it may be involved in later steps of cellulose degradation, thereby allowing the moss to make use of cellulose for energy production. Overall, our results suggest that the primordial role of BBE-like enzymes in plants revolved around primary metabolic reactions in carbohydrate utilization. DATABASE: Structural data are available in the PDB under the accession numbers 6EO4 and 6EO5.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions.

Biological oxidations form the basis of life on earth by utilizing organic compounds as electron donors to drive the generation of metabolic energy carriers, such as ATP. Oxidative reactions are also important for the biosynthesis of complex compounds, i.e. natural products such as alkaloids that provide vital benefits for organisms in all kingdoms of life. The vitamin B2-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) enable an astonishingly diverse array of oxidative reactions that is based on the versatility of the redox-active isoalloxazine ring. The family of FAD-linked oxidases can be divided into subgroups depending on specific sequence features in an otherwise very similar structural context. The sub-family of berberine bridge enzyme (BBE)-like enzymes has recently attracted a lot of attention due to the challenging chemistry catalyzed by its members and the unique and unusual bi-covalent attachment of the FAD cofactor. This family is the focus of the present review highlighting recent advancements into the structural and functional aspects of members from bacteria, fungi and plants. In view of the unprecedented reaction catalyzed by the family's namesake, BBE from the California poppy, recent studies have provided further insights into nature's treasure chest of oxidative reactions.