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. 2022 Dec 27;62(5):e202214400. doi: 10.1002/anie.202214400

Actinorhodin Biosynthesis Terminates with an Unprecedented Biaryl Coupling Reaction

Makoto Hashimoto 1,2,+, Susumu Watari 2,+, Takaaki Taguchi 1,3,+, Kazuki Ishikawa 1,2, Takuya Kumamoto 4, Susumu Okamoto 5, Koji Ichinose 1,2,✉
PMCID: PMC10108166  PMID: 36460615

Abstract

A plethora of dimeric natural products exist with diverse chemical structures and biological activities. A major strategy for dimerization is aryl coupling catalyzed by cytochrome P450 or laccase. Actinorhodin (ACT) from Streptomyces coelicolor A3(2) has a dimeric pyranonaphthoquinone structure connected by a C−C bond. In this study, we identified an NmrA‐family dimerizing enzyme, ActVA‐ORF4, and a cofactor‐independent oxidase, ActVA‐ORF3, both involved in the last step of ACT biosynthesis. ActVA‐ORF4 is a unique NAD(P)H‐dependent enzyme that catalyzes the intermolecular C−C bond formation using 8‐hydroxydihydrokalafungin (DHK‐OH) as the sole substrate. On the other hand, ActVA‐ORF3 was found to be a quinone‐forming enzyme that produces the coupling substrate, DHK‐OH and the final product, ACT. Consequently, the functional assignment of all essential enzymes in the biosynthesis of ACT, one of the best‐known model natural products, has been completed.

Keywords: Actinorhodin, Biaryl Coupling, Biosynthesis, Dimerizing Enzymes, Reaction Mechanisms

The last dimerization step of actinorhodin (ACT) biosynthesis was established through biochemical studies. ActVA‐ORF4 is a unique NAD(P)H‐dependent enzyme that catalyzes the biaryl‐coupling reaction using 8‐hydroxydihydrokalafungin (DHK‐OH) as the sole substrate. ActVA‐ORF3 is a quinone‐forming enzyme that forms DHK‐OH and ACT. The present results complete the functional assignment of essential enzymes for ACT biosynthesis.

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Introduction

Naturally occurring biaryl compounds display remarkable variations in structure, [1] even if restricted to those with benzenoid aromatic rings directly attached, ranging from a simple biphenyl neolignan, honokiol, [2] to a dimeric naphthylisoquinoline alkaloid, michellamine B with three biaryl linkages. [3] Axial chirality, resulting from the asymmetric substitution pattern on each side of the dimeric linkage, is another notable feature defining structural diversity and biological activities, as exemplified by a bridged glycopeptide, vancomycin whose biaryl configuration is exclusively the P isomer. [4] Enzymatic systems that govern regio‐ and stereoselective coupling reactions generating biaryl systems are of great interest. Representative examples of these enzymes are cytochrome P450s (CYPs) and laccases.[ 5 , 6 , 7 ] CYP‐based aryl coupling enzymes generate a series of reactive oxygen‐centered and carbon‐centered radicals, leading to the diradical connection in an inter‐ or intramolecular fashion. An aromatic resonance system gives rise to a variety of migrated radicals towards vast structural diversity in the coupled products. Well‐known biaryls generated by CYP dimerization reactions include anthraquinones, xanthones, naphthalenes, and naphthopyrones from a wide range of plants and microbes.[ 1 , 5 ] Laccases are another major class of dimerizing enzymes, which produce substrate phenoxy radicals, followed by divergent coupling reactions through C−C, C−O, and C−N bonds. Although laccases involved in lignan biosynthesis in higher plants had initially been characterized as nonspecific oxidases, their coupling reactions were shown to be stereoselective in the presence of dirigent proteins (DPs).[ 8 , 9 ] Biaryl‐producing laccases for fungal metabolites are more diverse in their mode of actions, as exemplified by the stereoselectivity directed by DP in viriditoxin biosynthesis [10] and the DP‐independent regiospecificity of the monapinone coupling enzymes. [11] Recently, a fasciclin‐domain‐containing (fas)‐protein was demonstrated to be required for the coupling activity as well as stereoselectivity in sporandol biosynthesis, and subsequent phylogenic analysis suggested a distinct group of laccase/fas protein systems in fungal phenol coupling reactions. [12] CYPs and laccases account for the major group of natural biaryls, whose coupling positions are mechanistically restricted to the ortho‐ or para‐position of the phenolic hydroxy group.

Pyranonaphthoquinone (PNQ) natural products such as kalafungin, [13] nanaomycin, [14] medermycin, [15] granaticin (GRA), [16] and dimeric PNQs[ 17 , 18 ] have been isolated from various organisms including bacteria, fungi, and plants. Actinorhodin (ACT, 1) from Streptomyces coelicolor A3(2) has a dimeric PNQ structure with connection of the halves by C−C bond at C10 (Figure 1). S. coelicolor A3(2) is the best‐known organism for its long history as a platform of Streptomyces genetics, and ACT has served as a model secondary metabolite[ 19 , 20 , 21 , 22 ] because of its characteristic blue pigmentation under basic conditions, enabling detection and quantification by visual inspection as well as spectrophotometric analysis. A unique feature of the ACT structure is that the C−C bond is established at the meta‐position of one of the hydroxy groups at the naphthazaline substructure in a regiospecific manner. [23]

Figure 1.

Figure 1

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The proposed biosynthetic pathway of actinorhodin.

The formation of the PNQ core for ACT biosynthesis is initiated by the formation of a C16 linear polyketomethylene intermediate from 8 malonyl‐CoA units by a type II minimal polyketide synthase (PKS) [24] following 6‐deoxy‐dihydrokalafungin (DDHK, 3) formation by several tailoring enzymes (Figure 1).[ 25 , 26 , 27 , 28 ] We have demonstrated the consecutive hydroxylation step from 3 to THK‐OH (hydroxy‐tetrahydrokalafungin, 7, equivalent to T4HN, 6) by a two‐component flavin‐dependent monooxygenase (FMO), ActVA‐ORF5/ActVB system[ 29 , 30 ] From the observation that the gene inactivation of actVA‐ORF4 results in accumulation of 7 and 8‐hydroxydihydrokalafungin (DHK‐OH, 8), [31] the last step in ACT biosynthesis was proposed to be the dimerization of a trihydroxynaphthalene derivative (T3HN, 4) through regiospecific aryl coupling catalyzed by the ActVA‐ORF4 protein. ActVA‐ORF4 resembles a family of nitrogen metabolite repression (Nmr) A proteins, functioning as transcriptional regulators. [32]

Our earlier studies[ 33 , 34 , 35 ] on the tailoring enzymes essentially required for dihydrokalafungin (DHK, 5) formation highlight actVA‐ORF3 as a functionally uncharacterized gene, which could be involved in a late stage of ACT biosynthesis. [36] This fact and the presence of its homologues in the biosynthetic gene clusters of GRA [37] and alnumycin [38] indicates that it is worth testing the possible involvement of ActVA‐ORF3 in the last stage of ACT biosynthesis (Figure S1).

Herein, we determined the enzymatic functions of ActVA‐ORF4 and ActVA‐ORF3, both indeed involved in the late stage of ACT biosynthesis. The dimerization reaction for ACT biosynthesis was revealed to proceed in sequence with the heteromeric connection between 6 and 8 by the actions of an iterative oxidase, ActVA‐ORF3 and a coupling enzyme, ActVA‐ORF4, thus presenting a unique example of biaryl formation in nature. Consequently, this study provides the final missing piece in ACT biosynthesis, thus allowing the elucidation of the complete biosynthetic pathway for a historically and scientifically important natural product, 1.

Results and Discussion

The fact that a ΔactVA‐ORF4 strain accumulated 7 and 8 in culture medium supports our proposal [31] that ActVA‐ORF4 is the dimerizing enzyme and 6 would be a substrate for the dimerization reaction. Therefore, the function of ActVA‐ORF4 was evaluated by in vitro assays using 7 or 8 as a substrate. Incubation of 7 with a recombinant ActVA‐ORF4 (Figure 2B and S2) yielded an unknown product, X, in the presence of NADPH as a cofactor, while 8 was detected in addition to 7 without ActVA‐ORF4 (Figure 2A), by autoxidation as previously reported. [31] Next, incubation of 8 with ActVA‐ORF4 yielded 1 together with X as a major product (Figure 2D). These results showed that the biaryl formation for ACT biosynthesis proceeds with ActVA‐ORF4 alone, and 8 is the true enzyme substrate.

Figure 2.

Figure 2

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In vitro analysis of aryl coupling activity for 7, 8 and 5 by ActVA‐ORF3 and ActVA‐ORF4. A) 7 without enzyme; B) 7 with ActVA‐ORF4 alone; C) 8 without enzyme; D) 8 with ActVA‐ORF4 alone; E) 5 without enzyme; F) 5 with ActVA‐ORF4 alone; G) 8 with ActVA‐ORF3 alone; H) 8 with ActVA‐ORF4 and ActVA‐ORF3.

In contrast, ActVA‐ORF4 did not accept 5 as a substrate (Figure 2E and 2F), suggesting its essential requirement for the 8‐hydroxy group for the aryl coupling activity. Furthermore, the presence of NADPH or NADH was found to be essential for ActVA‐ORF4 (Figure S3). LC‐MS analysis of X revealed the UV/Vis spectrum with λmax at 400 nm which was similar to that of THK‐OH. From a deprotonated ion ([M−H]−) at C32H29O14, the molecular composition of X was estimated as C32H30O14 which was 4 mass units higher than the molecular weight of 1 (Figure S4) and identical with that of the homodimer of 7, (hereafter named as THK‐OH dimer, 10, Figure 5). UPLC/Q‐TOF MSE analysis [39] was employed to elucidate further structural information on compound X. MS fragment patterns including dehydration, decarboxylation and a monomeric unit derived from 10 were successfully detected (Figure S4C). These results rigorously indicate that compound X is the 10, 10′‐linked dimer of 7. This fact indicates that the primary reaction product of ActVA‐ORF4 is the reduced form of 1. In addition, compound X was successfully detected in a wild type ACT producer, implying its in vivo relevance to ACT biosynthesis (Figure S4A). Although 10 is spontaneously oxidized to 1, a specific oxidase may accelerate this process.

The actVA‐ORF4 gene is co‐transcribed with the upstream gene actVA‐ORF3, whose function is currently unknown. To explore the function of ActVA‐ORF3, we expressed and purified an N‐His‐tagged version of the protein (Figure S2). ActVA‐ORF3 alone had no detectable enzymatic activity toward 8 (Figure 2G). Remarkably, an almost exclusive production of 1 was afforded in the assay mixture containing both ActVA‐ORF4 and ActVA‐ORF3 with 8 (Figure 2H). These results obviously indicate that the last stage of ACT biosynthesis consists of a sequence of reactions initiated with ActVA‐ORF4‐based coupling followed by the action of ActVA‐ORF3 to convert 10 to 1. Considering the structure of 10 equivalent to a reduced form of ACT (ACT‐red, 9, Figure 5), we assumed ActVA‐ORF3 to function as an oxidase for 10 and also its monomeric form, 7. Indeed, incubation of 7 only with ActVA‐ORF3 resulted in 8 production, indicating that ActVA‐ORF3 would also be involved before the dimerization step in ACT production (Figure S5). ActVA‐ORF3 shows no apparent homology with typical dehydrogenases, lacking a nucleotide‐binding motif. Omission of NADP+ from the assay mixture of ActVA‐ORF3 had no effect on its oxidase activity (Figure S5 and S6), suggesting ActVA‐ORF3 to be a cofactor‐independent oxidase. [40] We then investigated the possibility of an oxygen to be an electron acceptor based on the previous study [38] of Aln6, a distant ActVA‐ORF3 homologue (29 % identity) involved in the oxidation step for an unusual C−C bond formation in alnumycin biosynthesis. Indeed, we successfully detected a significant level of hydrogen peroxide derived through hydrogen abstraction to afford 8 and 1 from 7 and 10, respectively (Figure S7). [41]

To test whether actVA‐ORF3 is involved in ACT biosynthesis, the actVA‐ORF3 gene was inactivated in vivo. The resulting ΔactVA‐ORF3 strain with an empty vector (ΔactVA‐ORF3+pTYM18 [42] ) were fermented in R5MS medium and ACT‐related metabolite profiles were compared with that of the M510 strain, [43] an ACT producer (M510+pTYM18). LC‐MS analysis of their ethyl acetate extracts revealed that the ΔactVA‐ORF3+pTYM18 strain showed a drastic reduction in ACT production compared to the wild type strain (Figure 3). The reintroduction of the actVA‐ORF3 gene (ΔactVA‐ORF3+actVA‐ORF3) restored ACT production. These results imply that adverse effects of actVA‐ORF3 inactivation on ACT biosynthesis are not simply due to polar effect on the downstream genes. Indeed, the ΔactVA‐ORF3 strain retained the ability of 8 production, indicating that ActVA‐ORF3 would participate in ACT biosynthesis after the consecutive hydroxylations for 3 by ActVA‐ORF5/ActVB (Figure S8).

Figure 3.

Figure 3

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Gene inactivation studies of actVA‐ORF3 for 1 production. A) Effect of the ΔactVA‐ORF3 mutation on ACT production in Streptomyces coelicolor M510. Wild‐type (WT) and the mutant strain were grown on R5MS agar plates. B) LC‐MS analyses of a culture extract from S. coelicolor A3(2). 1) M510 strain (an ACT producer); 2) M510 with pTYM18 (an empty vector); 3) ΔactVA‐ORF3 with pTYM18; 4) ΔactVA‐ORF3 with actVA‐ORF3. Selected ion monitoring (SIM) at 633.1250 ([M−H]−) is shown.

To gain the mechanistic aspects in the coupling reaction, modeling and docking studies were conducted using a close ActVA‐ORF4 homologue, KstA11 (Figure 4, S9 and S10). KstA11 displays an overall similarity (48 % identity) with ActVA‐ORF4, with the available X‐ray crystallographic structure (PDB: 5F5N) complexed with a NAD+ (Template Ligand 1, TL1) and a 1,4‐dihydroxy‐anthracyline substrate (Template Ligand 2, TL2), [44] allowing us to perform a modeling study of ActVA‐ORF4. Homology modeling (HM) was conducted based on a Molecular Operating Environment (MOE) software platform. MOE‐Align was applied based on sequence and structural alignment for relevant proteins (Figure S9). HM was carried for the target proteins using an induced‐fit option with TL1‐derived NADH and TL2 to generate a reasonable model (Figure S10A and S10B). For the docking simulations, a modeled protein and NADH were defined as the receptor molecule, whereas a TL1 molecule was set for a docking site for the first ligand (DHK‐OH_1, L1) to generate a model binding NADH and L1 with the interactive distance within 4 Å (Figure S10C). A possible binding site was searched for the second ligand (DHK‐OH_2, L2) using Site‐Finder to generate dummy atoms representing the suitable spaces for L2. A reasonable ActVA‐ORF4 model complexed with NADH, and two molecules of 8 was obtained (Figure 4A, S10D and S10E).

Figure 4.

Figure 4

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Modeling study of ActVA‐ORF4 and Aryl coupling activity of the wild‐type and mutant ActVA‐ORF4 with ActVA‐ORF3. A) Docked model of ActVA‐ORF4 with ligand (DHK‐OH anion and NADPH). B) Putative interactions with amino acid residue of ActVA‐ORF4 and ligands (blue: DHK‐OH_1; magenta: DHK‐OH_ 2; orange: NADH). The distance between the pro‐S hydrogen at C4 of NADH and the C9 of DHK‐OH is shown by the red arrow (2.68 Å). C) Aryl coupling activity of ActVA‐ORF4 mutants compared to ActVA‐ORF4 wild type for ACT production from DHK‐OH.

After loading the first substrate (L1) molecule into ActVA‐ORF4, an oxygen atom at C8 of 8 acts as a hydrogen‐bond acceptor with Y262, and retains a series of residues (T14, D58, E90, and N154) interactive with NADH close to DHK_OH_1 (Figure 4B). In this model, the first substrate would be readily reduced by the hydride transfer from NADH in close proximity (2.68 Å, Figure 4B and S10E) to form T4HN, 6, which is favorably placed (3.48 Å, Figure S10E) near the second substrate molecule (L2) with a π‐π stacking interaction for the C−C bond formation between the C10 positions to take place.

From the resultant modeling structure of ActVA‐ORF4 using a singular substrate molecule, notable interactions were observed for the C8 oxygen atoms of the two substrates and tyrosine residues. Y262 would interact with the hydroxy group at C8 in one of 8 (L1) and Y254 with the other one (L2). The residues Y150 together with R173 would be implicated in the binding with the carboxyl group of 8 and appear to serve as hydrogen donor to hold the first substrate more tightly. In parallel, T14, D58, E90, K129 and N154 form a series of hydrogen bonds with the NADH molecule (Figure 4B).

The residues mentioned above were substituted to see if they are involved in the dimerization step. The Y262A and N154L mutant almost completely abolished the dimeric activity (Figure 4C, S11 and S12). The E90 A, K129A, R173E and Y254A mutant partially retained the activity for ACT formation. These results indicated that Y262 and N154 are essential for holding DHK‐OH and NADPH in the aryl‐coupling reaction. The 4 residues (E90, K129, R173 and Y254) would also be involved in the dimerization step.

The mechanistic outcome in silico was evaluated by a site‐directed mutagenesis study on in vitro assays of ActVA‐ORF4 to indicate that the Y262 of ActVA‐ORF4 is a critical residue for substrate recognition, and the hydroxy group at C8 of the substrate, 8, is required for the recognition of the PNQ skeleton on ActVA‐ORF4. Two of the residues (N154 and K129) suggested to interact with NADH were also proved to be essential for the dimerization activity. These results strongly support our mechanistic proposal.

Based on our results, the mechanism for the aryl coupling reaction in ACT biosynthesis would be as follows (Figure 5): the first molecule of the substrate 8 is reduced at C9 to 6′, which would function as a nucleophile to form a C−C linkage at C10′ of the second substrate 8. This is an unprecedented example in natural biaryl biosynthesis, particularly noteworthy being that the heteromeric connection between 6′ and 8 establishes the C−C bond and that ActVA‐ORF3, a cofactor‐independent oxidase, produces the coupling substrate (7 to 8, Figure S5) and the final product (10 to 1, Figure S6). The detection of hydrogen peroxide in the ActVA‐ORF3 assay made us propose its stepwise oxidation mechanism (Figure S13). Previously, a distinct mutant strain of S. coelicolor, mapped to actVA‐ORF3, was characterized [36] to produce only 5–10 % of blue pigments upon complementation with actVA‐ORF4, 5, and 6. This observation is explained by the critical role of ActVA‐ORF3 shown in this study (Figure 1).

Figure 5.

Figure 5

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Proposed mechanisms of aryl coupling reaction in ACT biosynthesis. The quinone‐forming step by ActVA‐ORF3 and the biaryl‐coupling step by ActVA‐ORF4 are highlighted in blue and green, respectively.

Conclusion

In this study, we have elucidated the final step of ACT biosynthesis, consisting of an unprecedented sequence of biaryl coupling reactions catalyzed by the two enzymes, ActVA‐ORF4 and ActVA‐ORF3. Our earlier studies [31] on a deletion mutant of actVA‐ORF4 of S. coelicolor led to the identification of 8, which was supposed to be an auto‐oxidized shunt product of 6/7 (Figure 1). This result allowed us to make a new proposal that a tetrahydroxynaphthalene derivative, 6, would function as a true substrate for the dimerization step, although its regioselectivity between C9 and C10 had remained to be clarified. [31] The intermediacy of 6/7 in ACT biosynthesis was recently proved [30] by our functional analysis of ActVA‐ORF5 and ActVB, catalyzing the consecutive hydroxylations at C6 and C8 of 3 to afford 7 (Figure 1).

We determined the enzymatic functions of ActVA‐ORF4 and ActVA‐ORF3, both involved in the biaryl coupling step of ACT biosynthesis. This is an unprecedented example of biaryl coupling in nature, consisting of an NmrA‐family dimerizing enzyme, ActVA‐ORF4 and a cofactor independent oxidase, ActVA‐ORF3. Two NmrA‐family proteins, Lom19, [45] and Strop2191, [46] both share over 50 % homology with ActVA‐ORF4, and are suggested to be involved in the C−C dimerization step of lomaiviticin biosynthesis, based on our earlier study [31] that THK‐OH and DHK‐OH was isolated from an ActVA‐ORF4 deletion strain. However, their biaryl‐coupling activity has not been experimentally proven. Apart from ours, an NmrA‐family enzyme for biaryl‐coupling reaction was only demonstrated at the in vivo level for nenestatin B. [47] A deletion strain of nes18 (47 % identity), similar to actVA‐ORF4, accumulates monomers of nenestatin B. [47] In the present study, we demonstrated in vitro for the first time that ActVA‐ORF4, an NmrA‐family enzyme, shows a unique biaryl‐coupling activity. The present results also allow us to complete the functional assignments of essential enzymes for each biosynthetic step of ACT biosynthesis, leading to an overall biosynthetic pathway to be elucidated for a historically and scientifically important natural product, 1.

Biosynthetic studies on ACT have brought us several surprising findings that revise the predictions derived from previous studies. An example is the functional assignment of actVB which had initially been proposed to function at the dimerization step based on the detection of the shunt product, kalafungin, by the S. coelicolor actVB mutant. [48] The function of ActVB is now established as a flavin reductase assisting ActVA‐ORF5 reductase as well as regulating the interconversion between the naphthoquinone (NQ) and hydronaphthoquinone (HNQ) forms of ACT intermediates. [30] Our previous characterization of ActVA‐ORF5/ActVB suggested an anticipated substrate for the dimerization to be a HNQ derivative 6/7, turning out not to be a substrate for the dimerization. In this study we have revealed that ActVA‐ORF3 comes to oxidize 6/7 to 8, which is to be subjected to the dimerizing step by, ActVA‐ORF4, followed by the second oxidation by ActVA‐ORF3. Thus, the later stages of the ACT biosynthetic pathway were found to proceed by enzymatic steps under the perfect control of the redox state of NQ and HNQ intermediates.

Conflict of interest

The authors declare no conflict of interest.

1.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

Click here for additional data file. (1.5MB, pdf)

Acknowledgments

We are grateful to Masaki Yabe for his assistance at the early stages of this work. We thank Aya Sawada, Faculty of Pharmacy, Musashino University for her assistance in experiments. We also thank Yoshirou Kimura, Molsis Inc. for helpful discussion. This work was supported by JSPS Kakenhi Grant Number 19H3385 (to KI) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Prof. Sir David Hopwood for critical reading of the manuscript.

Dedicated to Professor Sir David Hopwood

Hashimoto M., Watari S., Taguchi T., Ishikawa K., Kumamoto T., Okamoto S., Ichinose K., Angew. Chem. Int. Ed. 2023, 62, e202214400; Angew. Chem. 2023, 135, e202214400.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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