Biosynthetic Strategies of Berberine Bridge Enzyme-like Flavoprotein Oxidases toward Structural Diversification in Natural Product Biosynthesis

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon–carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.

N atural products exhibit astonishing structural diversity and this molecular variety results in a vast array of biological activities. 1 Nevertheless, secondary metabolites generally originate from a small number of starting components, which are obtained from primary metabolic pathways.−5 There are a multitude of biocatalysts involved in the modification of the metabolite precursors including oxidoreductases, halogenases, (acyl-, glycosyl-)transferases and ligases. 6n particular, oxidoreductases are intriguing since complex biochemical transformations often require changes in redox states. 7,8Therefore, oxidoreductases are essential players in the complex pathways that lead to the synthesis of secondary metabolites in different organisms.Oxidation reactions such as dehydrogenation, epoxidation and hydroxylation are typical redox reactions in natural product biosynthesis. 9Flavindependent oxidoreductases are able to perform these transformations and are therefore commonly observed in secondary metabolic pathways.This Review focuses on flavin-dependent oxidases from the vanillyl-alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) family. 10−13 VAO/PCMH Flavoprotein Family.Based on structural data and sequence homology there are six different families of flavin-dependent oxidases. 14One of these six families is the VAO/PCMH flavoprotein family, named after the fungal vanillyl-alcohol oxidase and bacterial p-cresol methylhydroxylase, feature a distinct flavin adenine dinucleotide (FAD) binding domain in the N-terminal portion of the protein. 12,15his conserved domain allows for the binding of the adenosine diphosphate and ribityl functional groups of FAD.Except for the flavin-binding domain, VAO-type enzymes contain a substrate-binding domain (cap domain) with greater variability positioned above the isoalloxazine ring of the cofactor.This structural arrangement enables significant diversity in the active site structures and, consequently, in the catalytic activities of these enzymes. 11−18 In the last few decades, many more members of the VAO family have been identified and shown to contain a covalently tethered flavin cofactor. 12Intriguingly, a significant number of them even had the flavin cofactor bicovalently bound to the protein, such as glucooligosaccharide oxidase (GOOX). 19,20−23 Another effect of covalently binding FAD is the increase in flavin redox potential, which is highest for bicovalently bound flavoproteins. 24Increasing the redox potential enhances the oxidative power of the flavin cofactor making them particularly adept at catalyzing demanding oxidation reactions.
Flavoprotein oxidases typically contain FAD, and sometimes flavin mononucleotide (FMN), as prosthetic group for mediating redox reactions. 9Reactions are catalyzed in a twostep manner, consisting of a reductive and oxidative half reaction (Scheme 1). 25 In the former half, the substrate is oxidized while the flavin is being reduced by hydride attack on the N5-atom.In the subsequent oxidative half reaction, the reduced flavin (Fl red ) returns to its oxidized state (Fl ox ) as the cosubstrate molecular oxygen (O 2 ) gets reduced.The first step in the oxidative half reaction is the single electron transfer of Fl red to O 2 producing a flavin semiquinone and superoxide species that can be covalently tethered yielding the C4ahydroperoxyflavin species (Fl 4aOOH ). 26This species can then undergo multiple dissociation pathways, leading to different reactivities, making flavins very versatile organic cofactors.Oxidases typically use elimination and proton transfer to release hydrogen peroxide (H 2 O 2 ) and thereby regenerate Fl ox .The elevated redox potential of (bi)covalently bound flavoprotein oxidases causes O 2 to be one of the few electron acceptors that they can employ.Hence, in order to regenerate the reduced cofactor, oxidases use O 2 as an electron acceptor by definition, effectively rendering the reaction irreversible.
Berberine Bridge Enzyme Subfamily.Within the VAO/ PCMH flavoprotein family, there are 11 different subfamilies out of which one has members involved in secondary metabolite biosynthesis in both plants and microorganisms. 23hese are named BBE-like enzymes and constitute a sizable portion of the VAO-fold oxidoreductase family.Apart from the common FAD-and substrate-binding domains, they have a distinct structural characteristic near the FAD-binding site which serves as a distinguishing feature of the BBE subfamily (Figure 1a). 27This structural characteristic has been annotated as a domain (pfam entry PF08031) and contains a special Cterminus with Y/FxN motif that, in the case of a Tyr, creates a specific hydrogen bonding network proximal to the isoalloxazine ring of FAD and conserved Asn residue (Figure 1b).This motif helps shape the O 2 binding pocket and affects the positioning of the ribityl moiety of FAD. 28he (S)-reticuline oxidase, commonly known as the berberine bridge enzyme (BBE), from the plant Eschscholzia californica is the name-bearer for this subfamily and catalyzes the conversion of (S)-reticuline to (S)-scoulerine by mediating an oxidative ring closure reaction (Scheme 2).This reaction is proposed to occur through a stepwise oxidation of the Nmethyl group to the iminium ion followed by a Friedel−Crafts acylation. 29(S)-Reticuline is the source of a wide range of benzylisoquinoline alkaloids (BIA) metabolites in secondary plant metabolism. 30,31A branch point in the biosynthesis of BIAs is marked by the C−C bond created by action of BBE, which is known as the berberine bridge. 32BBE-like flavoproteins typically contain a bicovalently bound FAD.−36 ■ UNCONVENTIONAL OXIDATION REACTIONS

CATALYZED BY BBE-LIKE OXIDASES
Even though BBE-like oxidases are named as such, they are functionally very different from the original name-bearer of this subfamily.They are capable of catalyzing a wide range of different reactions not only limited to the berberine bridge formation but also seen as essential enzymes for the generation of a plethora of intricate pharmacophores.−40 These BBE-like oxidases promote the rearrangement of the molecular skeleton by catalyzing carbon-heteroatom and carbon−carbon bond oxidations.Oxidation can lead to the formation of new intramolecular bonds or prime the substrate to undergo hydration or dimerization reactions.These redox reactions have been of widespread interest in the field of synthetic electrochemisty 41−43 and here we show the clever ways that nature utilizes BBEs to accomplish such challenging transformations.Even though BBE-like oxidases perform diverse activities, the exact mechanistic functioning of the enzymes performing the reactions is not always known.In this Review, we will go over multiple examples of experimentally validated BBE-like oxidoreductases in which the complexity of these biotransformations is portrayed.
Below we highlight four generalized modes of action by which BBE-like oxidases enable the synthesis of intricate natural products.In Scheme 3 an example reaction is given for each reaction type that BBE-like oxidases utilize in natural product biosynthesis.The first reaction type is classical alcohol oxidation, where hydride transfer occurs at the α-carbon atom.The example shown in Scheme 3a illustrates a simple alcohol oxidation causing the dienophile to become sufficiently electron deficient and therefore allowing a [4 + 2] cycloaddition. 44The second example is the deprotonation of a phenol-derivative which promotes the hydride transfer at a distant carbon atom (Scheme 3b). 45This is similar to the benzylic oxidation reaction catalyzed by the classic VAO, with the only difference being the ortho-orientation of the hydroxy group compared to the usual para-orientation.Hydride transfer is achieved via the generation of an ortho-quinone methide (ο-QM) intermediate, that can then undergo different reactions such as the [4 + 2] cycloaddition leading to THCA.This ο-QM intermediate is observed in many BBE-like oxidases some of which have already been discussed by Purdy et al. 46 The third example is the oxidation of a carbon− nitrogen bond, leading to hydride transfer from the α-carbon atom and thereby generating a transient imine or iminium cation that can undergo derivatization similar to the berberine bridge enzyme (Scheme 3c). 47The last example reaction is the hydride transfer from monoterpene indole alkaloids (Scheme 3d). 48The generated intermediate, stabilized via delocalized electrons from the indole N-atom, can undergo a multitude of derivatizations, including intramolecular cyclization but also water addition with concomitant oxidative cyclization.A phylogenetic tree was made but did not reveal clustering of the BBE-like enzymes into separate clades according to their function.The different activities might have evolved independently over time and other factors such as the enzyme's origin and the type of natural product produced could be a reason for this.
Alcohol Oxidation.Flavin-dependent oxidases are particularly known for the oxidation of alcohol groups.Examples   include the methanol oxidases from methylotrophic yeasts 49−51 and glucose oxidases 52 secreted by filamentous fungi.Below we elaborate on alcohol oxidations occurring in biosynthetic pathways that can facilitate subsequent noteworthy transformations.
The first example concerns a BBE-like oxidase found in Alternaria solani, a pathogenic fungus that causes early blight in tomato and potato plants. 53Numerous polyketides are produced by this fungus including solanapyrone, which has a decalin structure formed through a [4 + 2] cycloaddition comparable to lovastatin skeleton formation. 35,54Initially assuming that a polyketide synthase would be responsible for the cycloaddition reaction as seen with lovastatin, it turned out that the flavoprotein oxidase Sol5 was the enzyme involved. 44lthough this flavoenzyme performs a single oxidation of a primary alcohol group of prosolanapyrone II to the corresponding aldehyde, it also lowers the LUMO energy of the dienophile, thereby promoting the [4 + 2] cycloaddition (Scheme 4).Hence, the initial redox reaction performed by the BBE-like oxidase Sol5 is making this cycloaddition possible. 55other more recent example is a BBE-like oxidase involved in sorbicillinoid biosynthesis. 56Sorbicillinoids represent a substantial fungal natural product class encompassing over 100 derivatives characterized by intricate three-dimensional architectures and a wide spectrum of significant pharmacological activities. 57Sorbicillinoids are categorized into monomeric, dimeric, trimeric, and hybrid groups based on shared structural features.The biosynthetic pathway toward sorbicillinoids involves the collaboration of a highly reducing iterative polyketide synthase (hrPKS, SorA) and a nonreducing iterative polyketide synthase (nrPKS, SorB) to establish the core backbone of sorbicillin.Subsequently, an NAD(P)H-dependent flavoprotein monooxygenase SorC catalyzes the oxidative dearomatization to yield sorbicillinol.The last enzyme in the biosynthetic pathway is BBE-like oxidase SorD that has been shown to play a crucial role in the polymerization and oxygenation of sorbicillinol. 36,58,59Recently, SorD from Acremonium chrysogenum was heterologously expressed in Aspergillus nidulans to analyze its exact function. 56Sorbicillinol undergoes spontaneous hydration, as it is a highly reactive α,βunsaturated ketone generating the hydrated sorbicillinol Ansaseomycins are part of the macrolactam family and exhibit potent biological activities. 60The oxidoreductase AsmF was shown to be responsible for the oxidation of C23-OH of the nascent polyketide synthase product 1 to yield ketone compound 2 (Scheme 6).This paves the way, after keto−enol tautomerization, to a Diels−Alder reaction forming compound 3. 37 Knocking asmF out from the heterologous strain of Streptomyces seoulensis A01 led to an increase in novel ansaseomycin derivatives.Indeed, when the hydroxyl group was not modified by AsmF, the substrate could undergo spontaneous dehydration, forming 4, a common precursor for other deoxy-naphthalenic compounds.This observation implies that nature has developed a specific approach for incorporating the hydroxyl naphthalenic moiety in ansamycin natural products.Ultimately, the deletion of asmF might be an effective strategy to enhance the structural diversity of ansamycins.
AnuG was identified in the silent biosynthetic gene cluster involved in the oxidative lactonization forming the polyketide annullatin D. 61 Via overexpression in A. nidulans, it was shown to synthesize the five-membered lactone ring via the oxidation of the hydroxyl groups of the alkylated salicylaldehyde precursor (Scheme 7).It is postulated that the hydroxymethyl functional group undergoes a double oxidation to the acid. 61he first oxidation creates an aldehyde that, after the addition of water, forms a hydrate that can be oxidized to the acid.This is followed by spontaneous lactonization with the other hydroxyl group.−64 These oxidases are thought to be involved in the oxidation of the hydroxymethyl group to the aldehyde of a flavoglaucin congener, but they do not lead to the formation of five-membered lactones.This is because the flavoglaucin congener substrate lacks the hydroxyl group needed for intramolecular ring-closure.The cytochrome P450 monooxygenase (CYP) responsible for installing this hydroxyl group is not present in the BGC of N. crassa, and in A. ruber, the respective CYP hydroxylates the aromatic ring instead of the side chain.In T. virens, the CYP has not been characterized.
Another notable enzyme is BeklD, a bicovalently-FAD bound flavoprotein that is likely to enable epimerization of berkeleylactone derivatives by performing an alcohol oxidation. 65Berkeleylactones are macrocyclic polyketides produced by several Penicillium species. 66,67Even though it is postulated that a short-chain dehydrogenase/reductase (SDR) termed BeklG is responsible for the complete epimerization, it might instead be a combination of the SDR BeklG and oxidase BeklD (Scheme 8).Characterized homologues of BeklD have been found in other macrolide-producing organisms such as BerkD from Penicillium egyptiacum (96% seq id.) and ZEB1 from Fusarium graminearum (40% seq id.) where they perform a secondary alcohol oxidation. 66,68The ketone functional group could then be reduced into the other epimer by the SDR BeklG, thereby enabling epimerization and causing structural diversification.
A flavoprotein found in the marine-derived fungus Actinoalloteichus cyanogriseus WH1−2216−6 is noteworthy as it does not partake in the biosynthesis of the nonribosomal peptidepolyketide hybrid secondary metabolite caerulomycin A itself. 69,70This bicovalently FAD-bound oxidase named CrmK can recycle products back to the main biosynthetic pathway via a double oxidation reaction of an alcohol to a carboxylate via an aldehyde (Scheme 9).The on-pathway carboxylate substrate CRM O is transformed into the corresponding aldehyde intermediate CRM M using the dehydrogenase pair CrmN/CrmO.This aldehyde substrate is then stepwise converted to the Z-and E-configured aldoxime CRM H by the two-component monooxygenase CrmH.The unstable Z-CRM H, however, can spontaneously go back to the aldehyde CRMN M by a deoximation reaction.This would lead to an accumulation of the aldehyde intermediate.Since aldehydes are known for being reactive electrophiles, they are often detoxified by the cell enzymatically or spontaneously.In the cell, the aldehyde CRM M can spontaneously reduce to the corresponding alcohol CRM P in an inadvertent side reaction to potentially avoid buildup of the aldehyde intermediate.
However, CrmK can shunt it back to the aldehyde and, after hydrate formation with water, also oxidize it to the carboxylate CRM O although with lower efficiency.−73 Another flavoenzyme capable of a four-electron oxidation is called SapB from the sap cluster in Scedosporium apiospermum F41−1. 74This cluster encodes a PKS-NRPS (sapA) and flavindependent oxidoreductase (sapB) that together assemble a polyketide−amino acid (PKAA) conjugate type N-acylated amino acid (NAAA) that inhibits Arabidopsis root growth.Generally, the C-terminal domain of a fungal PKS-NRPS is reductive and can only release the amino-acyl adduct intermediate through a Dieckmann cyclization or Knoevenagel condensation making it impossible for a single fungal PKS-NRPS to produce a PKAA conjugate. 75Interestingly, SapA, after backbone formation, does not use such a release mechanism.Instead, it forms an N-acylated amino alcohol as an intermediate which can be used by the BBE-like oxidase SapB to perform a double oxidation creating the NAAA (Scheme 10).This work shows a new biosynthetic reasoning leading to a novel PKAA conjugate type NAAA enabled by combining a PKS-NRPS with a BBE-like oxidase.
The flavoprotein Dbv29 is the first described FMNcontaining bicovalent oxidase. 76Dbv29 is involved in the maturation of the glycopeptide A40926, a vancomycin-like glycopeptide, and an important antibiotic obstructing bacterial cell wall synthesis.By catalyzing two consecutive oxidation reactions, Dbv29 transforms the N-acyl aminoglucosamine into N-acyl aminoglucuronic acid (Scheme 11).Liu et al. 77 have been able to synthesize new antibiotic analogues for combatting antibacterial resistance by rationally intercepting the aldehyde intermediate and performing a chemoenzymatic reductive amination using different amines. 78nother unusual flavoenzyme is the aclacinomycin oxidase named AknOx.This bacterial-derived oxidase from Streptomyces galilaeus MA144-M1 is also capable of a four-electron oxidation but on two different functional groups: a − CH−OH and − CH 2 −CH 2 moiety. 79Aclacinomycins (Acl) are aromatic polyketides with antibiotic and antitumor activity, hence a compound of interest for disease treatments. 80Connected to Acl is a trisaccharide moiety of which the terminal L-rhodinose sugar residue is modified by AknOx (Scheme 12).The next biosynthetic step involves the elimination of two hydrogen A similar flavoenzyme named GcnQ (56% seq identity to AknOx) has been found in the grincamycin gene cluster of Streptomyces lusitanus. 81Grincamycin (GCN) has a tetragomycin skeleton containing a di-and trisaccharide substituent both with a terminal L-rhodinose moiety.Interestingly, in vitro this bicovalent FAD-containing GcnQ is able to perform the same double oxidation of the terminal sugar L-rhodinose to Laculose.It was shown that the oxidation and subsequent desaturation occur in tandem without forming the intermediate L-cinerulose A (Scheme 13), differently to what has been observed in aclacinomycin biosynthesis. 79Moreover, GcnQ has divergent roles when it is expressed in different hosts.In S. lusitanus SCSIO LR32, GcnQ manages to perform solely the single oxidation reaction of L-rhodinose to Lcinerulose A. However, when being heterologously expressed in S. coelicolor M512 it is able to transform the L-rhodinose units to L-aculose.This exemplifies the diverse activities that a single berberine bridgelike enzyme can exhibit in natural product biosynthesis.
TamL is a flavoprotein derived from Streptomyces sp.307−9 and involved in the biosynthesis of the antibiotic tirandamycin. 82This flavin-dependent oxidase displays intriguing enzymatic interplay with a cytochrome P450 monooxygenase (CYP) named TamI in effectively tailoring tirandamycin. 83he oxidative reaction occurs in a defined order, where TamI first performs hydroxylation of substrate TirC to TirE (Scheme 14).Thereafter, TamL must oxidize this C10 hydroxyl group to the corresponding ketone TirD.TirD is then again used a substrate for TamI converting it to TirA via epoxidation and subsequently to TirB by a final hydroxylation reaction.This iterative substrate exchange between TamL and TamI establishes a distinctive tailoring route wherein a CYP performs multiple oxidations in tandem with another biosynthetic enzyme.Together, these oxidative alterations within the tirandamycin pathway play a substantial role in enhancing its antibiotic potency toward inhibiting bacterial ribonucleic acid polymerase by increasing reactivity. 84,85nother bacterial-derived BBE-like oxidase is GilR from Streptomyces griseoflavus Go3592. 86This unusual lactoneforming oxidoreductase catalyzes the final step in the biosynthetic pathway toward the polyketide-derived gilvocarcin V. Gilvocarcin-type compounds show distinct anticancer activity through a light-mediated [2 + 2]-cycloaddition adduct with the side chain of DNA. 87,88GilR is responsible for oxidizing the hemiacetal to the lactone, thereby forming gilvocarcin V (Scheme 15).This lactone moiety is of crucial importance for the antibiotic's stability and potency.

Biochemistry
is the deprotonation of a phenolic moiety with subsequent or concomitant hydride transfer at a distant carbon atom.The aromaticity of phenolic hydroxyl groups enables the delocalization of electrons and the transfer of a hydride from a distant carbon atom to the N5-atom of FAD.This deprotonationassisted distant hydride transfer reaction can go via an ο-QM intermediate and multiple BBE-like oxidases have been hypothesized to employ this oxidation mechanism. 46he first example encompasses three BBE-like oxidases that are involved in the biosynthesis of the primary cannabinoids found in Cannabis sativa, 90−92 namely tetrahydrocannabinolic acid synthase (THCAs), cannabidiolic acid synthase (CBDAs) and cannabichromenic acid synthase (CBCAs). 45,93These three BBE-like oxidases act on the same precursor, cannabigerolic acid (CBGA), and share an oxidative carbon− carbon bond formation.Purdy et al. 46 proposed that the reaction is initiated by deprotonation of the C4 phenolic hydroxy group by a crucial tyrosine acting as a catalytic base (Scheme 16).This allows for subsequent or simultaneous transfer of the hydride to the oxidized FAD cofactor, generating the ο-QM intermediate.The following intra-molecular cyclization can go through different mechanisms and regioselectivities, generating the three different cannabinoids THCA, CBDA and CBCA.
Another BBE-like oxidase that is involved in oxidative cyclization via an ο-QM intermediate is Clz9 found in the BGC of Streptomyces sp.CNH-287 producing chlorizidine A. 94 This natural compound contains a pyrrolo-isoindolone ring, which is a new and unique structure in the field of natural products. 95Deprotonation of the phenolic hydroxyl moiety promotes hydride transfer from the benzylic C13 to FAD, forming an ο-QM intermediate.The enone is then attacked by the pyrrole's nitrogen creating chlorizidine A (Scheme 17a).A similar enzyme has been identified from Actinomycete strain AJS-327 where a homologous (53% seq.id.) FAD-dependent oxidoreductase was found and named Tcz9. 38The oxidation reaction is again proposed to include an ο-QM intermediate but instead of a nucleophilic attack of pyrrole's nitrogen, it forms a dichloropyrrole-containing compound featuring an α,β-unsaturated ketone (Scheme 17b).
ο-QM intermediate can also be found in the secondary metabolic pathway of mulberry plants (Morus alba). 33Once a

Biochemistry
plant gets infected by a fungus, it produces chalcomoracin as a mean to protect the leaves via fungal germination suppression. 96This flavonoid is formed through an enzymatic Diels− Alder reaction between a diene (the isoprenyl portion of an isoprenylphenol) and a dienophile (double bond of morachalcone A).Interestingly, there are two BBE-like enzymes involved in this specific biosynthetic pathway and they show high (50%) sequence identities with THCAs, CBDAs and CBCAs. 45 cyclization of the formed diene with the dienophile morachalcone A, representing the first known stand-alone intermolecular Diels−Alderase (Scheme 18b).MaDA will be discussed in more detail in the later section "Other Mechanisms".
Two closely related BBE-like oxidases, PenH and AsqF, perform an atypical oxidation-mediated extension of the prenyl side chain in the biosynthesis of the penigequinolone and aspoquinolone alkaloids of Penicillium thymicola and A. nidulans. 97−99 A base-catalyzed reaction was proposed in which the C4 proton is removed and the C1 hydride is transferred to the N5 atom of the FAD cofactor. 40However, based on recent research of ο-QM intermediates, it might be more plausible that proton abstraction of the C6 phenolic alcohol promotes hydride transfer (Scheme 19).Both  102 Another peculiar BBE-like oxidase is ElcE involved in the biosynthesis of the aromatic polyketide perylenequinone elsinochrome A found in Parastagonospora nodorum.ElcE was found to be responsible for the coupling of two nahpthol derivatives, requiring a laccase-like multicopper oxidase ElcG. 103The same was observed in Cercospora nicotianae, where the ElcG homologue, CTB12, was deleted thereby completely abolishing cercosporin production. 104By heterologous biosynthesis, it was demonstrated that both ElcE and ElcG are needed for the double coupling step, yielding the pentacyclic perylenequinone (Scheme 21).As laccases are known for catalyzing radical coupling reactions, 3 it was suggested that ElcG catalyzes the first coupling via a phenol  103 However, the exact mechanism has yet to be elucidated.
Amine and Amide Oxidation.Many BBE-like enzymes are effective amine oxidases that generate transient imines or iminium cations that can undergo derivatization.The namebearer of the BBE-like subfamily catalyzes this type of oxidation reaction (Scheme 2). 23A concerted mechanism was proposed where deprotonation of the C3-phenol, hydride transfer from the N-methyl group to the FAD and ring closure occur in a single step. 27However, a later-performed study on the solvent and substrate deuterium kinetic isotope effect suggested that an earlier proposed stepwise mechanism would be more likely. 29,105In the stepwise mechanism, a hydride is transferred from the N-methyl group forming a methylene iminium ion intermediate (Scheme 22).Thereafter, the activesite base Glu417 deprotonates C3-phenol, making the C2 carbon more nucleophilic and able to attack the N-methylene moiety, forming the berberine bridge.
A similar amine oxidase involved in benzylisoquinoline alkaloid biosynthesis is the (S)-tetrahydroprotoberberine oxidase (STOX) from Berberis wilsoniae. 106,107STOX can oxidize C−N bonds of the protoberberine alkaloid tetrahydroprotoberberine to produce palmatine (Scheme 23). 108oreover, STOX has high sequence similarity to the BBE name-bearer from E. californica with 38%.Unsurprisingly, BBE itself also displays STOX activity although with lower efficiency. 109A four-electron oxidation reaction has to take place to synthesize palmatine and the initial oxidation forming the iminium ion is thought to proceed enzymatically and the second oxidation to proceed spontaneously. 106Mechanistic investigations concluded that the iminium ion was formed between C14 and N7, creating an unstable intermediate that can undergo spontaneous oxidation in the presence of O 2 .
Another example involves the monoterpenoid indole alkaloids catharanthine and tabersonine, produced by the plant Catharanthus roseus.These alkaloids are intermediates in the biosynthesis of the potent anticancer drugs vincristine and vinblastine and a BBE-like amine oxidase is involved in the synthesis of the scaffolds. 110This BBE-like enzyme was named precondylocarpine acetate synthase (PAS) and together with dihydroprecondylocarpine synthase (DPAS), tabersonine synthase (TS) or catharanthine synthase (CS) act in a concerted manner to produce tabersonine and catharanthine (Scheme 24). 34In vitro analysis showed that PAS oxidizes the C−N bond of stemmadenine acetate producing the iminium intermediate precondylocarpine acetate, which is subsequently reduced by DPAS and undergoes [4 + 2] cyclization by TS or CS.Interestingly, PAS lacks the His and Cys residues necessary for the FAD cofactor's covalent attachment.Homologues of PAS, sharing amino acid identities ranging from 68% to 74%, have been identified in other Apocynaceae plant species, suggesting their potential involvement in the assembly of similar monoterpene indole alkaloids through a conserved mechanism. 110 peculiar BBE-like enzyme named FsBBE has been found in the biosynthetic pathway of plant-derived Securinega alkaloids that enables intermolecular enamine-type addition reactions. 111The substrate allosecurinine is oxidized by FsBBE to form the iminium ion and tautomerization generates the enamine intermediate (Scheme 25).The C3 atom can then act as a nucleophile and attack the C2 of L-ascorbic acid (or its oxidative product dehydroascorbic acid).This enamine-type addition reaction enables a spontaneous cascade reaction of ketal formation and cyclization to fluesuffine A. Previously, it was proposed that many C2 or C3-funtionalized Securinega alkaloids were produced via a key enamine intermediate which acts as a branching point for further derivatization. 112,113In The flavoprotein SfmCy2 makes use of a transient iminium ion to accomplish the oxidative deamination forming the natural product saframycin A. 114 SfmCy2 is expressed extracellularly by bearing a Tat signal peptide and allows for prodrug-maturation of saframycin A in Streptomyces lavendulae by catalyzing a final deamination reaction.This reaction proceeds through dehydrogenation of the amino group to form an imine intermediate (Scheme 26).The resulting imine is attacked by water to form the hemiaminal, which after NH 3 release forms the ketone.The reaction was also performed in H 2

18
O and afforded an enzymatic product with a molecular weight increase of 2 Da as compared to the reaction performed in H 2 16 O.This validates that the ketone group is originating from water.This study uncovers another unique activity that BBE-like enzymes exhibit, namely, oxidative deamination.
Another secreted BBE-like protein from Streptomyces bearing a Tat signal peptide is named NapU and performs an oxidative activation and overoxidative inactivation of the matured prodrug naphthyridinomycin (NDM). 115The first reaction that NapU catalyzes is the hydroxylation of the pharmacophore inactivated compound 1 to form the bioactive NDM (Scheme 27).When the reaction mixture was incubated for a longer period of time, another product 2 was formed with a decrease in molecular weight of 2 Da that did not show antibacterial activity.The first hydroxylation reaction is proposed to go through the reactive iminium intermediate.H 2 O may act as the nucleophile, potentially activated by two Tyr active site residues, to attack the imine and install the hydroxy-group in NDM.It was illustrated that NapU can also mediate the subsequent two-electron oxidation, yielding the corresponding inactivated ketone-derivative.Hence, the flavoprotein NapU is involved in the activation of a matured prodrug, but can also avoid self-toxicity via overoxidation of NDM as self-defense strategy.
Although there is a considerable number of identified FADdependent amine oxidases, 116 amide oxidases are not commonly known in literature.They are predicted to be involved in the biosynthesis of certain fungal-derived natural products such as pyranonigrin and pyranterreones. 117,118The first study that fully characterized a BBE-like oxidase performing an unprecedented amide oxidation is FmqD, which operates in the biosynthesis of two cytotoxic peptidyl alkaloids, fumiquinazoline C (FQC) and D (FQD). 47,119hese metabolites are a key feature of the pathogenic fungus A. f umigatus and have received considerable attention due to their complex biochemistry. 120Thanks to co-ordination with sporulation-specific transcription factors, FmqD is secreted to the cell wall and directs its product to the fungal spores. 120mqD primes its so-called substrate fumiquinazoline A (FQA) for C−O and C−N intramolecular cyclization.Specifically, amide oxidation leads to a transient imine, which is captured by the −OH group of the imidiazoindolone side chain, producing the spirohemiaminal FQC (Scheme 28). 47Slow equilibration ultimately forms the aminal FQD as a more thermodynamically stable product.The absence of the lone-pair stabilization for the amide might seem to make hydride abstraction of the α-carbon challenging, but it is speculated that the extended delocalized system of the FQ scaffold manages to stabilize the cation to allow for hydride abstraction.This example illustrates the delicate chemistry required for amide oxidation, leading to regioselective product cyclization.
Monoterpene Indole Alkaloids.A few BBE-like enzymes oxidize an isoprenyl moiety, typically conjugated to an indolederived scaffold and also called monoterpene indole alkaloids.The mechanism by which this occurs has not yet been understood, and some propose the hydride transfer to occur at the benzylic C10 atom assisted via abstraction of the C13 hydrogen or vice versa.Taking into account the other BBE-like oxidation mechanisms discussed so far, we propose that the hydride transfer is enabled by the electron donating effect of the N1 atom through the indole ring to the C10 atom (Scheme 29).This ultimately allows the hydride to be transferred to the N5 atom of FAD.Such a mechanism would be similar to the one of the ο-QM intermediate. 46The obtained reactive iminium ion intermediate can undergo a multitude of derivatizations including intramolecular cyclizations, dehydrogenations, and hydration reactions.

Biochemistry
The first example can be found for ergot alkaloids, which are prenylated indole alkaloids produced by several filamentous fungi and have wide applications in therapeutics due to their similarity with monoamine neurotransmitters such as dopamine and adrenaline. 121−123 A BBE-like oxidase involved in the biosynthesis of ergot alkaloid D-lysergic acid is EasE, a bicovalent flavoenzyme from the parasitic fungus Claviceps purpurea.Together with a catalase partner enzyme EasC, EasE is involved in the oxidative cyclization of 4-dimethylallyl-Labrine (4DMA) to chanoclavine-I (Scheme 30). 124These two enzymes are essential for the central carbon ring in the tetracyclic ergoline core structure.Until recently, it remained an enigma how this central C ring was formed. 39Essentially, EasE performs a dehydrogenation reaction after which the O 2dependent catalase EasC can complete the cyclization via a radical addition mechanism.
The FAD-dependent oxidoreductase CnsA and putative catalase CnsD from Penicillium expansum share homology to EasE and catalase partner EasC (51 and 59% seq.id.). 48,125,126hese enzymes operate on 4-L-dimethyl-allyl tryptophan that is oxidized at the isoprenoid unit.However, subsequent cyclization is not caused by a radical addition mechanism but rather by an intramolecular attack of the α-NH 2 group (Scheme 31).This leads to trans-clavicipitic acid, which forms aurantioclavine after decarboxylation by the putative catalase CnsD.

Biochemistry
Another type of meroterpenoids is the indole diterpenoids synthesized by various fungi.This meroterpenoid class lacks the polyketide scaffold and instead possesses an indole ring as nonterpenoid part. 127Indole diterpenes are generally synthesized via a common hexacyclic molecule named paspaline. 128his natural product class obtains variability through branching out using different biosynthetic gene clusters. 129,130ere, we will give an overview of three different FADoxidoreductases present in the biosynthetic pathways producing the three indole diterpene natural products shearinines, penitrems, and nodulisporic acids.
−133 JanO was analyzed in vitro and yielded a single product, suggesting that cyclization goes rapidly without intermediate product formation.Incubating the reaction with 30% H 2 O 18 clarified that the O 2 atom of the cyclic ether ring was originating from water. 132Thus, JanO catalyzes a cascade of reactions: oxidation of the isoprenyl group, hydration, another oxidation, and cyclization yielding the final shearinine B product.Recently, the ortholog of JanO named NodO (52.1% seq id.) was confirmed to be responsible for the oxidative cyclization forming nodulisporic acid D 4 . 131ost likely the nodulisporic acid D 4 synthesis goes via a similar cyclization pathway. 134,135he third oxidoreductase PtmO from Penicillium simplicissimum 136 catalyzes a similar reaction in that its substrate's isoprenyl group is oxidized, enabling the incorporation of a hydroxyl group coming from water (Scheme 33).The diversity of these reaction illustrates how indole diterpenoids can be used for generating a diverse set of molecules. 130her Mechanisms.All of the reactions described so far all have an overarching mechanism in which a hydride is transferred to the N5 atom of the FAD cofactor.Nevertheless, examples of BBE-like oxidoreductases can be found that do not employ the FAD cofactor for catalysis.Moreover, there are examples in which the role of the FAD cofactor is still unclear.

Biochemistry
The first example is the Diels−Alderase named MaDA, 33,137 already briefly mentioned during the biosynthesis of morachalcone A in mulberry plants (Scheme 18b).After oxidation of the isoprenyl moiety of moracin C by the BBE-like oxidase MaMO, MaDA enables a [4 + 2] cycloaddition reaction making it the first stand-alone intermolecular Diels− Alderase.Multiple controls were performed to understand the role of the FAD cofactor in MaDa, including mutagenesis of the His116 residue that is critical for covalent FAD attachment and cofactor reduction using sodium dithionite.This mutation rendered MaDA almost inactive, thereby exemplifying that the (oxidized) FAD cofactor is necessary for the endo [4 + 2] cycloaddition.Moreover, homologous Diels−Alderases were discovered that could catalyze the same intermolecular Diels− Alder reaction with exo-selectivity. 138Mutagenesis studies demonstrated key residues involved in activating the dienophile through hydrogen bonding interactions and positioning the diene via π−π interactions for endo-selectivity.While for opposite selectivity a crucial arginine residue forms a cation−π interaction with the dienophile, which lowers the free energy barrier for the exo-pathway and thereby regulates selectivity.The evolutionary origin of MaDA and MaMO was also investigated and suggested their Diels−Alderase and oxidative dehydrogenation activity to have originated from a gene duplication and subsequent neofunctionalization of an oxidocyclase. 139Nevertheless, the exact function of the FAD cofactor in MaDA remains ambiguous.
Another example is the enzyme SthB, involved in the biosynthesis of the phytotoxic polyketide stemphyloxin II. 140A highly similar (74% seq.id.) enzyme has been found in betaenone B and C-producing Phoma betae. 141The striking aspect about SthB is that it catalyzes a stereoselective aldol reaction to form a bridged tricyclo[6.2.2.0 2,7 ]dodecane scaffold (Scheme 34).The intramolecular aldol reaction is hypothesized to proceed via the standard base-catalyzed aldol reaction that is nonoxidative.SthB allows for a proton abstraction thereby producing the nucleophilic enolate that attacks the C1carbonyl on the decalin ring, forming stemphyloxin II.No hydride is being transferred to the N5 atom of the FAD and the role (if any) of the cofactor in SthB has remained unexplored, pending additional biochemical characterization. 142nother peculiar BBE-like oxidase is called AspoA, which is involved in the biosynthesis of cytochalasans, fungal polyketide-nonribosomal peptides with antibiotic properties (PK-NRPs). 143,144Interestingly, this flavoenzyme from Aspergillus f lavipes does not perform a dehydrogenation reaction but acts as general acid biocatalyst to catalyze double bond isomer- ).Addition of FAD did not increase activity, and deletion of His158, which covalently tethers the cofactor, did not decrease activity.A site-directed mutagenesis study was performed to identify the crucial residues and showed that Glu538 was highly conserved as mutagenesis caused a halt in activity.Isotope labeling studies confirmed that the double bond isomerization proceeds via protonation of the C21 carbonyl group, a hydride shift, and a keto−enol tautomerization.AspoA homologues such as ffsJ (85% seq.id.) from A. flavipes 145 and PhmC (60% seq.id.) from P. nodorum 146 might potentially perform similar reactions as they both harbor the conserved glutamate residue.
A distinctive flavoenzyme named PylE was recently found in the biosynthetic gene cluster of Setosphaeria sp.SCSIO41009, responsible for the synthesis of pyrenophorol dilactones. 147imilar to AspoA (30% seq id), this flavoprotein also catalyzes an isomerization reaction. 143In this case, PylE catalyzes the isomerization of the 4-alcohol-2,3-unsaturated moiety of pyrenophorol, producing a 1,4-diketone.Two mechanisms were hypothesized for the isomerization reaction (Scheme 36).In the first mechanism, it was proposed that oxidation of the C4 hydroxyl occurs first, after which a hydride is transferred from FAD to C3 to create the enol intermediate.Tautomerization then leads to the first ketone product.Another isomerization round would then yield the final 1,4diketone product.The second mechanism goes via initial protonation of the C1 carbonyl, after which the C4 proton is abstracted to form the enol intermediate.Tautomerization and subsequent isomerization of this intermediate create the 1,4diketone product.Mutagenesis studies proved acid−base residue Glu526 to be a crucial residue for catalytic activity.With the help of docking, the authors showed this residue to be in the vicinity of the C1 carbonyl group and therefore could function as a general acid and base in route b.In contrast to AspoA, mutating the conserved His153 necessary for covalent tethering of the FAD cofactor abolished PylE's activity.This would suggest its involvement in isomerase activity, but a complete mechanistic study is yet to be performed.
For a long time, oxygenation reactions were commonly thought to be catalyzed by FAD-dependent monooxygenases using the Fl 4aOOH species. 9Nevertheless, in 2013, the BBE-like enzyme named EncM was shown to feature a different oxygenating species termed as the flavin-N5-oxide (Fl N5O ). 148,149This oxygenase is part of a BGC in Streptomyces maritimus, forming enterocin, an unusual polyketide antibiotic.The Fl N5O species triggers a Favorskii rearrangement by simultaneous hydroxylation and dehydrogenation (Scheme 37), reminiscent of the postredox cyclization reactions such as Sol5. 35,55So far, EncM has been the only enzyme employing the flavin-N5-oxide intermediate for catalysis within the BBElike subfamily of oxidoreductases.Nevertheless, this exception sparked the investigation on the versatility of the N5 position and expanded the repertoire of known flavin catalyzed reactions. 150

■ CONCLUSION
The BBE-like oxidases from the vanillyl-alcohol oxidase/pcresol methyl hydroxylase flavoprotein family are fascinating enzymes exhibiting extraordinary chemical activities.The ability to transfer a hydride atom to the FAD cofactor enables complex substrate modifications and rearrangements, including intramolecular cyclizations, Diels−Alder reactions, Michael additions, and hydroxylations.This leads to a structurally and functionally diverse number of natural products in bacteria, plants, and fungi.A mechanistically recurrent theme of most of these enzymes is the ability to elicit molecular skeleton rearrangements triggered by an initial oxidation step through hydride transfer to the FAD.However, there are also cases in which the exact involvement of the FAD cofactor still remains unknown or in which the FAD cofactor utilizes a different oxygenating species.There is a plethora of uncharacterized BBE-like enzymes, and genomic context is needed to predict their functions.Understanding the natural mechanisms that are employed to generate the incredible diversity of natural products is a topic of great interest in biochemistry for its fundamental implications in the field.Hence, the study and characterization of these new enzymes will allow us to uncover more unique biochemical activities.

Scheme 1 .
Scheme 1. Reductive and Oxidative Half Reaction of Flavoprotein Oxidases a

Figure 1 .
Figure 1.(a) The structure of EcBBE (PDB: 3D2J) with the FAD-domain in silver, the substrate-domain in dark sea green and the BBE-domain in moccasin.The FAD-cofactor is colored orange, and all atoms are shown as ball-and-stick models.(b) The FAD-binding site of EcBBE featuring the distinctive C-terminal Y/FxN motif.The FAD cofactor is bicovalently bound to residues Cys166 and His104.

Scheme 2 .
Scheme 2. Oxidative Ring Closure Reaction from (S)-Reticuline to (S)-Scoulerine Catalyzed by the Plant-Derived Berberine Bridge Enzyme a

Scheme 4 .
Scheme 4. [4 + 2] Cyclization Enabled by the Alcohol Oxidation of Prosolanapyrone II to Aldehyde Mediated by the FAD-Enzyme Sol5

Scheme 5 .
Scheme 5. (a) The NAD(P)H-Dependent Flavoprotein Monooxygenase SorC Installs a Hydroxy-Group in Sorbicillin Producing the Reactive Sorbicillinol a and (b) the Reactive Sorbicillinol Undergoes a Michael-Addition Reaction with Oxosorbicillinol to Produce the Dimer Acresorbicillinol C

Scheme 10 .
Scheme 10.Four-Electron Oxidation Performed by SapB to Form an N-Acyl Amino Acid in Scedosporium apiospermum F41-1 a Scheme 13.Oxidation Reactions Catalyzed by GcnQ In Vitro Producing Different Grincamycin Derivatives Depending on the Host Organism and Reaction Conditions

Scheme 16 .
Scheme 16.Deprotonation-Assisted Distant Hydride Transfer of Cannabigerolic Acid (CBGA) to Form an ο-QM Intermediate a First, the BBE-like moracin C oxidase (MaMO) oxidizes the isoprenyl moiety of moracin C into the diene (Scheme 18a).The authors suggest a similar ο-QM intermediate in which deprotonation of the aromatic hydroxy group of moracin C leads to hydride transfer and subsequent diene formation via tautomerization.Next, the second BBElike enzyme named Diels−Alderase (MaDA) enables [4 + 2]

Scheme 18 .
Scheme 18.(a) The BBE-like Oxidase MaMO Catalyzes the Oxidation of the Isoprenyl Moiety of Moracin C via an ο-QM Intermediate and (b) the Second BBE-like Oxidase MaDA Catalyzes the [4 + 2] Cyclization of the Formed Diene with Morachalcone A to Generate the Natural Product chalcomoracin

Scheme 20 .
Scheme 20.Oxidative Pyran Ring Formation Thought to Be Catalyzed via an ο-QM Intermediate a

Scheme 25 .
Scheme 25.Intermolecular Enamine-Type Addition Reaction Catalyzed by the Plant-Derived FsBBE via an Initial Amine Oxidation

Scheme 32 .
Scheme 32.(a) Hypothesized Reaction Mechanism of JanO Allowing for the Double Oxidation of the Isoprenyl Side-Group Thereby Forming the Secondary Metabolite Shearinine B and (b) Oxidative Cyclization Reaction of NodO Producing Nodulisporic Acid D 4

Scheme 34 .
Scheme 34.Stereoselective Aldol Reaction Synthesizing Stemphyloxin II is Hypothesized to Go via a Nonoxidative Base-Catalyzed Aldol Reaction in SthB