66NISR Research GRANT
2005 Young Investigator Research Grant

Production of Polyketides by a Novel P-450 Aryl Coupling
Catalyst
Nobutaka FUNA
Graduate School of Agriculture and Life Sciences, The University of Tokyo

Research aims
The genus Streptomyces comprises Gram-positive, soildwelling, filamentous bacteria with a complex life cycle
similar to that of fungi. In addition to the complex morphological differentiation, Streptomyces is also characterized
by its ability to produce a wide variety of secondary
metabolites including pharmaceutically useful compounds,
such as antibiotics, anti-tumor agents and immunosupressants. We have recently characterized a P-450 gene, named
P-450mel, which is responsible for 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (HPQ) synthesis in S. griseus.
In this report, we describe in vitro reconstitution of the P450mel catalyst with spinach ferredoxin-NADP reductase/ferredoxin. P-450mel catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which
was formed from five molecules of malonyl-CoA by the action of RppA1), to yield HPQ. We then investigated the substrate specificity of P-450mel. P-450mel accepted flaviolin
as a substrate to give dimmers of flaviolin, whereas several
dihydroxy naphthalanes and emodin anthrone were not accepted under the condition used in this study.
Methods
Production and purification of P-450mel. The nucleotide sequence (TGACGA) covering the TGA stopcodon of P-450mel was changed to CTCGAG to create an
XhoI site by PCR with primer I: 5-GCGAAGCTTCATATGGAGAACACCTCG-3 (the italic letters indicate a HindIII site, the underline indicates an NdeI
site, and the boldface letters indicate the start codon of P450mel) and primer II: 5-GCGGAATTCCTCGAGCCATGTGACGGGC-3 (the italic letters indicate an EcoRI site
and the underline indicates an XhoI site). The amplified
1.2-kb fragment was cloned between the HindIII and EcoRI
sites of pUC19, resulting in pUC19-P-450mel-Xho. The
NdeI-XhoI fragment excised from pUC19-P-450mel-Xho
was cloned between the NdeI and XhoI sites of pET26b, resulting in pET26b-P-450mel.
For production of P-450mel with a His-tag at its C-terminus, E. coli BL21 (DE3) harboring pET26b-P-450mel wasgrown at 37°C for 2 h in M9 medium containing 100 mM
FeSO4 and 100 m g/ml of ampicillin, followed by addition of80 m g/ml of 5-aminolevulinic acid hydrochloride and 100m M isopropyl b -D-thiogalactopyranoside (IPTG). After further incubation at 22°C for 1 day, the cells were collectedby centrifugation. A crude cell-lysate was prepared by soni-cation and removal of cell debris by centrifugation at
10,000 g for 30 min. P-450mel was purified by using a Ninitrilotriacetic acid column (Qiagen), according to the manual from the manufacturer. Protein concentrations weremeasured with a Bio-Rad protein assay kit using bovine
serum albumin as a standard. The sodium dithionate-reduced carbon monoxide difference spectrum of the purified
P-450mel was measured by the method described by
Omura and Sato2).
In vitro reconstitution of P-450mel activity. The reactions, containing 100 mM sodium phosphate (pH 7.3), 1mM dithiothreitol, 1 mM EDTA, 10% glycerol, 1 mMNADPH, 0.5 U of spinach ferredoxin:NADP reductase, 40m g spinach ferredoxin, and 23.4 m g of P-450mel, 400 m Mof THN, were performed in a total volume of 500 m l. Ferredoxin and ferredoxin:NADP reductase from spinach werepurchased from Sigma. THN was synthesized according tothe method of Ichinose et al.3). The reactions were carriedout at 30°C for 30 min and terminated by adding 50 m l of 6M HCl, and extracted with 200 m l of ethylacetate. The organic layer was collected and evaporated and the residualmaterial was dissolved in 20 m l of methanol for HPLC
analysis. Conditions of HPLC are as follows: ODS-80Ts
column (4.6150 mm, Tosoh), maintained at 40°C, waseluted with 25 mM KH2PO4 (pH 4.7) containing 13%
CH3CN at a flow rate of 1 ml/min.
Results
In vitro analysis of aryl coupling of THN by P-450mel.
For convenient purification of P-450mel, we placed its coding sequence under the control of the T7 promoter in
pET26b and introduced it into E. coli BL21 (DE3). Plasmid
pET26b-P-450mel thus constructed would direct the synthesis of P-450mel-Leu-Glu-His6. P-450mel purified fromthe soluble fraction using histidine-bind resin gave a single
protein band of about 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. P-450mel produced in E.
coli was an active form, because the purified enzyme had
an absorbance at 450 nm on binding CO (results notshown).
For reconstitution of the P-450mel activity for in vitro
enzyme assay, we used the ferredoxin and ferredoxinNADP reductase from spinach, as was successfully used
for reconstitution of a soluble cytochrome P-450soy from S.
griseus4). Incubation of the reconstituted system with THN
gave two products, as analyzed by HPLC (Fig. 1A). The
product at retention time 9 min was identified as HPQ by its

67Fig. 1. HPLC analysis of in vitro products. A, The products from THN
by the reconstituted P-450mel system were analyzed by simultaneously
measuring the absorbances at 254 nm (in red) and 440 nm (in black). B,
Authentic HPQ prepared from S. lividans harboring pNF16) was coinjectedwith the products from the P-450 reaction on THN. C, As a negative control, P-450mel was boiled before use for the reaction of P-450mel on
THN. D, As a negative control, ferredoxin and ferredoxin-NADP reductase were removed from the reaction. E, The products from pseudoHPQ by
the P-450mel reaction were analyzed. The peaks indicated with asterisks,derived spontaneously from pseudoHPQ were not identified. F, As a negative control, P-450mel was boiled before use for the reaction on
pseudoHPQ.

Fig. 2.

Proposed HPQ melanin biosynthetic pathway in S. griseus.

co-migration with authentic HPQ, which was preparedfrom S. lividans harboring pNF1 (Fig. 1B). The product at
retention time 6.2 min appeared to be pseudoHPQ (Fig.
3A), a dimeric form of THN, because it had a molecularmass of 382, which is equal to [2Mw2] Da of THN (Mw192), as revealed by liquid chromatography-atmosphericpressure chemical ionization mass spectrometry analysis(data not shown). The in vitro formation of HPQ from THN
by the action of P-450mel was consistent with the in vivo
observation that simultaneous overexpressison of rppA andP-450mel in S. lividans led to accumulation of HPQ.
Insignificant amounts of HPQ and pseudoHPQ were observed in the control incubations with a boiled enzyme(Fig. 1C) and without the redox-partners (Fig. 1D), indicating that these products were also formed non-enzymaticallyas a result of free radical-induced oxidation of THN, but ata lesser rate. We then performed the reaction usingpseudoHPQ, which was recovered from the reaction ofTHN, as a substrate to clarify whether both steps of sequential oxidation of THN leading to HPQ were catalyzed by P450mel. P-450mel rapidly consumed pseudoHPQ to yield
HPQ (Fig. 1E), although pseudoHPQ was extremely unstable and underwent spontaneous intramolecular aryl coupling to yield HPQ in a control reaction containing a boiled
P-450mel enzyme (Fig. 1F). These results suggest that P450mel catalyzes sequential oxidation of THN, which is intermolecular aryl coupling of THN and intramolecular aryl
coupling of resultant pseudoHPQ, to yield HPQ (Fig. 2).
Substrate specificity of P-450mel. The aryl coupling activity of P-450mel toward several aromatic compounds wasinvestigated. P-450mel did not reacted with 1-naphthol, 2naphthol, 1,3-dihydroxynaphthalene (1,3-DHN), 1,4-DHN,1,5-DHN, 1,6-DHN, 1,7-DHN, 2,3-DHN, 2,6-DHN, 2,7DHN, emodin, and emodin anthone. In contrast, P-450mel
reacted readily with flaviolin to give dimmers of flaviolin.
Interestingly, we have recently found that MomA, whichconstitutes an operon with RppA and P-450mel, catalyzes
oxidation of THN to give flaviolin5). This finding suggests
that, in some Streptomyces other than S. griseus, flaviolin,
but not THN, is the physiological substrate of P-450mel.
Taken all together, our results suggest that P-450mel possesses a strict specificity toward physiological substrates,
THN and flaviolin.

68Conclusion

References

The present study has demonstrated that P-450mel mediates the biaryl coupling of THN without inserting an oxygen atom into the product, HPQ. The reaction mechanism isbest explained by a radical coupling, as follows. One electron oxidation of THN affords a phenolate radical and subsequent intermolecular radical coupling furnishes the arylaryl bond in a regiospecific manner. Resultant pseudoHPQis subjected to further intramolecular aryl coupling thatelaborates HPQ (Fig. 2).

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