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A NOVEL ORTHO-AMINOPHENOL OXIDASE RESPONSIBLE FORFORMATION OF PHENOXAZINONE CHROMOPHORE OF GRIXAZONE

Hirokazu Suzuki, Yasuhide Furusho, Tatsuichiro Higashi, Yasuo Ohnishi,and Sueharu Horinouchi¶

From the Department of Biotechnology, Graduate School of Agriculture and Life Sciences,University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Running title: o-Aminophenol Oxidase Catalyzing Phenoxazinone Formation¶ To whom correspondence should be addressed: Department of Biotechnology, Graduate School of Agricultureand Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: 81-3-5841-5123; Fax: 81-3-5841-8021; E-mail: asuhori@mail.ecc.u-tokyo.ac.jp

Grixazone, containing a phenoxazinonechromophore, is a secondary metabolite producedby Streptomyces griseus. In the grixazonebiosynthesis gene cluster, griF encoding a tyrosinasehomologue and griE encoding a protein similar tocopper chaperons for tyrosinases are encoded.Expression study of GriE and GriF in Escherichiacoli showed that GriE activated GriF bytransferring copper ions to GriF, as is observed for aStreptomyces melanogenesis system in which MelC1copper chaperon transfers Cu ions to MelC2tyrosinase. In contrast with tyrosinases, GriFshowed no monophenolase activity, although itoxidized various o-aminophenols as preferablesubstrates rather than catechol-type substrates.Deletion of the griEF locus on the chromosomeresulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and itsacetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBALto yield an o-quinone imine derivative, which wasthen non-enzymatically coupled with anothermolecule of the o-quinone imine to form aphenoxazinone. Co-existence of N-acetylcysteinein the in vitro oxidation of 3,4-AHBAL by GriFresulted in the formation of grixazone A, whichsuggests that the –SH group of N-acetylcysteine isconjugated to the o-quinone imine formed from 3,4-AHBAL, and the conjugate is presumably coupledwith another molecule of the o-quinone imine.GriF is thus a novel o-aminophenol oxidase that isresponsible for the formation of the phenoxazinonechromophore in the grixazone biosyntheticpathway.

We have long studied the A-factor regulatorycascade that leads to secondary metabolite formationand morphological differentiation in Streptomycesgriseus (1, 2). A-factor (2-isocapryloyl-3R-hydroxymethyl-g-butyrolactone) triggers the synthesisof almost all the secondary metabolites produced by thisspecies. One of the secondary metabolites under the

control of A-factor is grixazone. Grixazone is a yellowpigment and actually a mixture of grixazone A and B (1aand 1b; see Fig. 2C) (3). Grixazone A was a novelcompound and grixazone B was reported to show aparasitcide activity (4). Grixazones contain a phenoxazinone chromophore.The phenoxazinone skeleton is common to actinomycin Dproduced by Streptomyces antibioticus (5), michigazoneproduced by Streptomyces michiganensis (6), texazoneproduced by Streptomyces sp. WRAT-210 (7), exfoliazoneproduced by Streptomyces exfoliatus (8), and 4-demethoxymichigazone produced by Streptomyceshalstedii (9). Hsieh and Jones (10) reported aphenoxazinone synthase (PHS1) in S. antibioticus thatcatalyzed the 6-electron oxidative coupling of o-aminophenol compounds derived from tryptophanthrough 3-hydroxyanthranilic acid. However, disruptionof the PHS gene in S. antibioticus did not affectactinomycin D synthesis, showing that the phenoxazinoneskeleton in actinomycin D is biosynthesized in vivo by astill unknown enzyme or non-enzymatically (11). On theother hand, michigazone with a hydroxymethyl group atthe 8-position of the phenoxazinone and texazone with acarboxyl group at this position were assumed to besynthesized from a precursor(s) different from that ofactinomycin D with no functional group at this position (7).No study on the precursor(s) or the biosynthetic enzymefor the phenoxazinone skeleton of these compounds has sofar been reported. Because grixazone A and B contain analdehyde and a carboxyl group, respectively, at the 8-position, the biosynthesis of the phenoxazinone skeleton ingrixazones should be the same as those of michigazoneand texazone. The phenoxazinones of exfoliazone and 4-demethoxymichigazone, both containing a hydroxymethylgroup at this position, are also presumed to be synthesizedin a similar way. We cloned the grixazone biosynthesis gene clusterand revealed the organization of the genes (ourunpublished data). Since all the biosynthesis andregulatory genes for a certain secondary metabolite areusually contained within the gene cluster, we expected thatone of the gene products encoded in this gene cluster was

http://www.jbc.org/cgi/doi/10.1074/jbc.M505806200The latest version is at JBC Papers in Press. Published on November 10, 2005 as Manuscript M505806200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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responsible for the formation of the phenoxazinoneskeleton of grixazones. Because some tyrosinasesshow an additional activity to use o-aminophenol as asubstrate and yield phenoxazinones (12, 13), we aimedat two gene products, named GriE and GriF, that aresimilar to MelC1 and MelC2 proteins in streptomycetes,respectively. MelC2 is a tyrosinase and MelC1 is acopper chaperon for MelC2. Tyrosinase is a copper-containing monooxygenase that catalyzes two-types ofreaction, the o-hydroxylation of monophenols(monophenolase activity) and the oxidation of o-diphenols to o-quinone (diphenolase activity) usingmolecular oxygen. Tyrosinase is a member of thetype-3 copper protein family, which also includeshemocianins and catechol oxidases. The type-3copper proteins contain a binuclear copper active sitethat is composed of two closely spaced copper atoms,each coordinated by three histidine residues. Althoughthe binuclear copper active site is highly conserved, asdetermined by its characteristic spectroscopic signatures,by sequence homologies, and by the crystal structuresof several hemocianins and a catechol oxidase, theirfunctions in catalysis are different. Catechol oxidasesoxidize diphenols to the corresponding quinones butlack tyrosine hydroxylase activity. Hemocianins serveas oxygen carriers and oxygen storage proteins inarthropods and mollusks. Tyrosinases are responsiblefor melanin pigmentation in streptomycetes, browningin plants, and melanization in animals. In addition tothe intrinsic tyrosinase activities, the tyrosinases fromNeurospora crassa (12), mushroom (13), andStreptomyces glaucescens (12) convert 2-aminophenolsinto the corresponding o-quinone imines in vitro at alesser rate, which are then non-enzymatically convertedinto phenoxazinones. In the present study, we determined the plausiblesubstrate of GriF by structural elucidation of anintermediate (3-amino-4-hydroxybenzaldehyde; 3,4-AHBAL; 2a, see Fig. 2C) that was accumulated in agriEF-deleted mutant and revealed the in vitroformation of a phenoxazinone skeleton from 3,4-AHBAL by recombinant GriF produced in Escherichiacoli. GriF did not use tyrosine as a substrate, althoughit showed about 50% identity in amino acid sequence tomelanin-producing tyrosinases in Streptomyces. Wehave thus concluded that GriF is an o-aminophenoloxidase responsible for the in vivo formation of thephenoxazinone chromophore in the grixazonebiosynthetic pathway.

EXPERIMENTAL PROCEDURES

Bacterial strains, media, and materials – S. griseusIFO13350 was obtained from the Institute of

Fermentation, Osaka, Japan. S. griseus strains weregrown at 26.5°C or 30°C in YPD medium (0.2% yeastextract, 0.4% Bacto peptone, 0.5% NaCl, 0.2%MgSO4·7H2O, 1% glucose, and 0.5% glycine; pH 7.2) andSMM medium (0.9% glucose, 0.9% asparagine, 0.2%(NH4)2SO4, 0.24% Trizma base, 0.1% NaCl, 0.05% K2SO4,0.02% MgSO4·7H2O, 0.01% CaCl2, 0.0034% (0.25 mM)KH2PO4, and 1% trace element solution (14); pH 7.2). Athiostrepton resistance plasmid pIJ702, with its copynumber of 40 to 300 (14), was used for expression of griR.Escherichia coli JM109 and TOP10 (Invitrogen) andplasmids, pUC19 and pCR4Blunt-TOPO (Invitrogen),pET-17b, and pET-26b, were used for DNA manipulation.E. coli BL21 (DE3)/pLysS was used for production ofGriE and GriF. Restriction enzymes, T4 DNA ligase,Pyrobest DNA polymerase and other DNA-modifyingenzymes were purchased from Takara Biochemicals.DNA was manipulated in Streptomyces (14) and E. coli(15) as described earlier. L-Tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), and benzoiccompounds except for 3-amino-4-hydroxybenzaldehyde(3,4-AHBAL) were purchased from Wako Chemicals.3,4-AHBAL was purified from the culture supernatant of S.griseus mutant DgriEF harboring pIJ702-griR (see below).The tyrosinase from mushroom was purchased fromSigma-Aldrich.

Deletion of griEF – The chromosomal griEF region of S.griseus IFO13350 was deleted as follows (see Fig. 1A).A 3.6-kb NcoI-SphI fragment containing a regionupstream from griE and a 3.6-kb PmaCI fragmentcontaining a 3’ portion of griF and a region downstreamfrom griF were connected with a short linker derived fromthe multi-cloning linker of pUC19. This 7.2-kb fragment,which had a 1.1-kb deletion of the sequence in the griEFlocus (from Met-3 of GriE to His-226 of GriF), was clonedin pUC19 together with a 1.1-kb HindIII fragmentcarrying the kanamycin resistance gene from Tn5,resulting in pDgriEF. The plasmid was alkali-denaturedand introduced by protoplast transformation into S. griseusIFO13350. Kanamycin-resistant transformantscontaining pDgriEF in the chromosome, as a result of asingle crossover, were first isolated and one of thetransformants was cultivated several times on YPDmedium without kanamycin. Kanamycin-sensitivecolonies, derived after a second crossover, were candidatesfor griEF-disrupted strains (mutant DgriEF). The correctreplacement was checked by Southern hybridization byusing a 0.5 kb PmaCI-SphI fragment as 32P-labeled probe(see Fig. 1A).

Identification of 3,4-AHBAL and 3,4-AcAHBAL in thesupernatant of S. griseus mutant DgriEF – For the purposeof overproduction of grixazone and its intermediates, we

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placed griR under the control of the melC promoter inpIJ702. The griR-coding sequence was amplified bythe following two primers, 5’-GCGCATGCCTACGCACCATC-3’ (the italic lettersindicate the start codon of griR and the underlinedletters indicate an SphI site) and 5’-TCGAATTCTCAGCCGGAGCGCCC-3 (the italicletters indicate the stop codon of griR and theunderlined letters indicate an EcoRI site). After theamplified fragment had been cloned in pCR4Blunt-TOPO and sequenced to confirm the absence of PCR-generated errors, the griR sequence was excised withSphI plus EcoRI and cloned between the SphI andEcoRI sites of a pIJ702-derived plasmid, in which themelC1-melC2 sequence was replaced by a short linkercontaining SphI, BglII, and EcoRI sites, resulting inpIJ702-griR. S. griseus mutant DgriEF containing pIJ702-griR was cultivated at 26.5°C with rotary shaking for 3days in SMM medium supplemented with 10 mg/mlthiostrepton. The culture broth was cleared bycentrifugation at 6,000 x g for 20 min and treated withhexane for liquid-liquid distribution. The materialsin the aqueous layer were extracted with ethylacetateand dried by evaporation. The crude material wasdissolved in dimethylsulfoxide (DMSO) and appliedto reversed-phase HPLC using a Waters 600 HPLCsystem equipped with a Waters 996 photodiode arraydetector. Compounds, 2a and 2b, were collected.HPLC conditions: column, Capcell Pak C18 (10 x250 mm, Shiseido Fine Chemicals); flow rate, 3ml/min; solvent A, 0.1% (v/v) trifluoroacetic acid inwater; solvent B, 0.1% (v/v) trifluoroacetic acid in90% (v/v) acetonitrile in water. After 100 ml of thecrude material had been injected into the columnequilibrated with 5% B, the column was initiallydeveloped isocratically for 3 min, followed by elutionby a linear gradient from 5% B to 100% B in 13 min.3,4-AHBAL (2a) and 3,4-AcAHBAL (2b) wereidentified, on the basis of the following spectroscopicparameters (see Fig. 2C). 3,4-AHBAL (2a): 1HNMR (500 MHz, DMSO-d6) d 9.63 (s, 1H, CHO), d7.10 (d, 1H, J = 2.5 Hz, ArH), d 7.03 (dd, 1H, J = 8.0,2.5 Hz, ArH), d 6.81 (d, 1H, J = 8.0 Hz, ArH); 13CNMR (125 MHz, DMSO-d6) d 191.6 (C-7), d 150.7(C-4), d 137.6 (C-3), d 129.3 (C-1), d 122.4 (C-6), d114.0 (C-2), d 112.5 (C-5); high resolutionelectrospray ionization-time-of-flight mass spectrum,m/z 137.09312 (M)+ (calculated for C7H7N1O2, 2.39mmu error). 3,4-AcAHBAL (2b): 1H NMR (500MHz, DMSO-d6) d 11.12 (s, 1H, OH), d 9.76 (s, 1H,CHO), d 9.34 (s, 1H, NH), d 8.41 (d, 1H, J = 2.0 Hz,ArH), d 7.53 (dd, 1H, J = 8.5 Hz, 2.0, ArH), d 7.03 (d,1H, J = 8.0 Hz, ArH), d 2.12 (s, 3H, CH3); 13C NMR

(125 MHz, DMSO-d6) d 191.3 (C-7), d 169.2 (C-8), d153.6 (C-4), d 128.3 (C-3), d 127.7 (C-6), d 127.2 (C-1),d 122.6 (C-2), d 115.4 (C-5), d 23.8 (C-9); high resolutionelectrospray ionization-time-of-flight mass spectrum, m/z180.06632 (M+H)+ (calculated for C9H10N1O3, 0.25 mmuerror).

Expression of griE and griF in E. coli – The griF sequencewas amplified by PCR with two primers, 5’-CATATGGTCCACGTACGCAAG-3’ (the italic lettersindicate the start codon of griF and the underline indicatesan NdeI site) and 5’-CTCGAGCTGGTCGTAGGTGTAGAAC-3 (theunderline indicates an XhoI site). After the amplifiedfragment had been cloned into pCR4Blunt-TOPO andsequenced to confirm the absence of errors during PCR,the griF sequence was excised with NdeI and XhoI andcloned into pET-26b(+). The griF sequence, togetherwith the lacI gene, was excised from the plasmid bydigestion with Bpu1102I and Bst1107I and transferredonto pET-17b digested with Bpu1102I and Bst1107I,resulting in pET-griF which contained griF under thecontrol of the T7lac promoter. The griE sequence wasamplified by PCR with primers, 5’-CATATGCCCATGAACAGGCGAG-3’ (the italic lettersindicate the start codon of griE and the underline indicatesan NdeI site) and 5’-CTCGAGTCAGAGGTGCGTGACGTG-3 (the italicletters indicate the stop codon of griE and the underlineindicates an XhoI site). The amplified fragment wascloned into pCR4Blunt-TOPO and sequenced to confirmthe absence of errors during PCR. The griE sequencewas excised with NdeI and XhoI and cloned in pET-17b,resulting in pET-griE which contained griE under thecontrol of the T7 promoter. For construction of plasmidcontaining both griE and griF, pET-griF was digested withSphI and treated with T4 DNA polymerase to produceblunt ends, followed by BglII digestion. To this linearplasmid with a BglII site at one end and a blunt end at theother end, a 0.9-kb fragment containing the griEexpression cassette excised from pET-griE by digestionwith SspI and BglII was ligated, resulting in pET-griEFwhich contained griE under the control of the T7 promoterand griF under the control of the T7lac promoter. Theseplasmids were used to transform E. coli BL21(DE3)/pLysS cells. Transformant cells were cultured at26.5°C for 10 h in LB medium supplemented with 50mg/ml ampicillin, 34 mg/ml chloramphenicol, and 1%(w/v) lactose allowing constant expression of the T7 andT7lac promoters.

Purification of recombinant GriF – All operations werecarried out at 4°C. Cells (3.6 g wet weight) wereharvested by centrifugation and suspended in 6 ml of

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buffer A (50 mM sodium phosphate, pH 8.0, 0.5 MNaCl, and 10% glycerol) containing 10 mM imidazoleand 2 mg/ml lysozyme. After incubation of themixture on ice for 30 min, the cell suspension wassonicated for 3 min and then centrifuged at 10,000 x gfor 10 min to remove cell debris. Polyethyleneiminewas added to the supernatant to give a finalconcentration of 0.1% (w/v), followed by centrifugationof the mixture at 20,000 x g for 20 min. Thesupernatant was a crude enzyme solution used for someanalyses. For further purification of GriF, thesupernatant was applied to a column (1 ml) of Cu2+-charged HiTrap Chelating HP (Amersham) equilibratedwith buffer A containing 10 mM imidazole. Thecolumn was successively washed with buffer Acontaining 10 mM imidazole and buffer B (50 mMsodium phosphate, pH 8.0) containing 50 mMimidazole, and then the materials were eluted withbuffer B containing 200 mM imidazole. The eluatewas applied to a HiTrap Q column (Amersham)equilibrated with buffer B containing 200 mMimidazole, and the column was washed with the samebuffer. The flowed-through and washing fractionswere pooled and ammonium sulfate was added to give20% saturation. The enzyme solution was applied toFPLC equipped with a Resource PHE column (1 ml;Amersham) equilibrated with buffer C (50 mM sodiumphosphate, pH 7.2, 20%-saturated ammonium sulfate)and eluted at a flow rate of 1 ml/min. The column waswashed with the same buffer for 10 min, followed byelution by a 0-100% linear gradient of buffer D (50 mMsodium phosphate, pH 7.2, 50% (v/v) ethylene glycol)in 10 min. Proteins were quantified by using a Bio-Rad Protein Assay kit with bovine serum albumin as thestandard. Gel filtration analysis was performed usinga Superdex 200 10/30 column (Amersham) on FPLCwith isocratic elution in 20 mM sodium phosphate,adjusted to pH 7.2, containing 0.15 M NaCl at a flowrate of 0.2 ml/min.

Enzyme assays – The standard reaction mixture (400 ml)consisted of 50 mM sodium phosphate, pH 7.0, 5 mMsubstrate, and an enzyme sample. The mixturewithout enzyme was preincubated at 30°C in a cuvetteand the reaction was started by the addition of theenzyme. GriF activity was assayed by monitoring theformation of 2-aminophenoxazin-3-one (APO; e433,9600 M-1 cm-1) from o-aminophenol as a substrate bymeasuring the absorbance at 433 nm on a spectrometer(SPECTRA MAX plus; Molecular Devices). Theinitial velocity was used for the calculation of activity,which was defined as the amount (mol) of substratedecreased per second at 30°C. Tyrosinase activity ofGriF and the mushroom tyrosinase was assayed by

monitoring the formation of dopachrome (e475, 3600 M-1

cm-1) from L-tyrosine or L-DOPA as a substrate bymeasuring the absorbance at 475 nm (16). One unit ofGriF activity was defined as the amount of enzyme thatdecreases 1 nmol substrate per second at 30°C．

Identification of the reaction products from o-aminophenol,o-aminophenol plus catechol, and 3,4-AHBAL – Theproducts were analyzed by reversed-phase HPLC.Conditions for HPLC: column, Senshu Pak DOCOSIL-B(4.6 x 250 mm, Senshu Kagaku); column temperature,30°C; flow rate, 1 ml/min. After 10 ml of the reactionmixture had been injected into the column equilibratedwith 0% B, the column was initially developedisocratically for 3 min, followed by elution with a lineargradient from 0% B to 100% B in 15 min. The reactionproducts from o-aminophenol, o-aminophenol pluscatechol, and 3,4-AHBAL were identified as APO (3a, seeFig. 2C), HPO (3b), and 2-aminophenoxazin-3-one-8-aldehyde (3c, APOAL), respectively, on the basis of thefollowing spectroscopic parameters. APO (3a): 1H NMR(500 MHz, DMSO-d6) d 7.69 (dd, 1H, J = 8.0, 1.5, ArH), d7.49 (dd, 1H, J = 8.3, 1.5 Hz, ArH), d 7.45 (td, 1H, J = 7.0,1.5 Hz, ArH), d 7.38 (td, 1H, J = 7.5, 1.5, ArH), d 6.36 (s,1H, ArH), d 6.35 (s, 1H, ArH); 13C NMR (125 MHz,DMSO-d6) d 180.2(C-3), d 149.1 (C-10a), d 148.1 (C-4a),d 147.6 (C-2), d 141.9 (C-5a), d 133.4 (C-9a), d 128.8 (C-7), d 127.7 (C-9), d 125.3 (C-8), d 116.0 (C-6), d 103.5 (C-1), d 98.1 (C-4); high resolution electrospray ionization-time-of-flight mass spectrum, m/z 213.06874 (M+H)+

(calculated for C12H9N2O2, 2.34 mmu error). HPO (3b):1H NMR (500 MHz, DMSO-d6) d 7.81 (d, 1H, J = 7.0,ArH), d 7.59 (t, 1H, J = 7.5 Hz, ArH), d 7.54 (d, 1H, J =8.0 Hz, ArH), d 7.44 (t, 1H, J = 7.5, ArH), d 6.69 (s, 1H,ArH), d 6.43 (s, 1H, ArH); 13C NMR (125 MHz, DMSO-d6) d 180.3(C-3), d 155.7 (C-2), d 149.1 (C-10a), d 148.6(C-4a), d 142.6 (C-5a), d 133.2 (C-9a), d 131.1 (C-7), d128.9 (C-9), d 125.5 (C-8), d 116.2 (C-6), d 106.7 (C-1), d104.2 (C-4); high resolution electrospray ionization-time-of-flight mass spectrum, m/z 214.06998 (M+H)+

(calculated for C12H8N1O3, 4.25 mmu error). APOAL(3c): 1H NMR (500 MHz, DMSO-d6) d 10.05 (s, 1H,CHO), d 8.20 (d, 1H, J = 2.0 Hz, ArH), d 7.93 (dd, 1H, J =8.5, 1.5 Hz, ArH), d 7.66 (d, 1H, J = 8.0, ArH), d 6.43 (s,1H, ArH), d 6.36 (s, 1H, ArH); 13C NMR (125 MHz,DMSO-d6) d 191.8 (CHO), d 180.5 (C-3), d 149.2 (C-10a),d 148.6 (C-4a), d 147.8 (C-2), d 146.0 (C-5a), d 133.9 (C-9a), d 133.2 (C-8), d 130.1 (C-7), d 128.1 (C-9), d 117.1(C-6), d 104.3 (C-1), d 98.2 (C-4); high resolutionelectrospray ionization-time-of-flight mass spectrum, m/z241.09174 (M+H)+ (calculated for C13H9N2O3, 30.42 mmuerror).

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RESULTS

GriF as a tyrosinase homologue – We cloned andsequenced the whole gene cluster for the biosynthesis ofgrixazone from S. griseus IFO13350 (Fig. 1A; ourunpublished data). The gene cluster contained 13open reading frames. Of the 13 gene products, twoproteins, named GriE and GriF, were similar to MelC1and MelC2 proteins, respectively, both of which areresponsible for melanin biosynthesis in streptomycetes(17-21). MelC2 tyrosinase belongs to the type-3copper protein family and MelC1 serves as a copperchaperon for MelC2. Although tyrosinases fromvarious organisms from bacteria to human havedifferent secondary and tertiary structures and domainstructures, they all contain two Cu-binding sites, eachformed by three histidine residues (Fig. 1B). The twoCu-binding sites of GriF are also highly conserved.Therefore, GriF was expected to contain two Cu atoms.The two closely spaced Cu atoms, each coordinated bythree histidine residues, must comprise a binuclearcopper active site of GriF, as is found for the type-3copper proteins. Thus, because of the high end-to-endsimilarity (about 50% identity) to MelC2, GriF wasexpected to show monophenolase and/or diphenolaseactivity by the same catalytic mechanism as tyrosinasesdo by using molecular oxygen as an oxidizing agent. GriE shows end-to-end similarity to MelC1 instreptomycetes and has a probable twin-argininetranslocation (TAT)-type NH2-terminal signal peptidefor protein secretion (data not shown), as does MelC1(22). The amino acid sequences of MelC1 instreptomycetes are conserved at a moderate level (40 to50% identity) and always encoded as a neighbor ofmelC2 (17, 20). GriE was therefore expected to beinvolved in the transfer of Cu ion to the apo-form ofGriF via binary complex formation (23, 24).

Accumulation of 3,4-AHBAL and 3,4-AcAHBAL in S.griseus mutant DgriEF – Because some tyrosinasesshow the ability to convert o-aminophenols into thecorresponding phenoxazinone moieties, in addition totheir intrinsic tyrosinase activity (12, 13), we expectedthat a mutation in griF would result in accumulation ofan o-aminophenol(s) as a substrate of GriF for theformation of the phenoxazinone chromophore ofgrixazone. We deleted the griEF locus on thechromosome of S. griseus IFO13350 to determine thechemical structure of a possible substrate for GriF, or anintermediate in the grixazone biosynthetic pathway (Fig.1A). As a result of double reciprocal crossover, a 1.1-kb griEF locus, corresponding to the region from Met-3of GriE to His-226 of GriF, was deleted, which wasconfirmed by Southern hybridization with the 0.5 kb

PmaCI-SphI fragment, shown in Fig. 1A, as 32P-labeledprobe against the chromosomal DNA digested with SphI(data not shown). HPLC analysis of the culture broth of mutantDgriEF revealed the accumulation of some compoundsdifferent from grixazones. After 3 days cultivation,mutant DgriEF accumulated two major compounds, 2aand 2b, that were not seen in the culture broth of theparental strain, when detected by absorbance at 254 nm(Fig. 2A and B). After 5 days cultivation, the parentalstrain accumulated grixazone A (1a), when detected byabsorbance at 433 nm, characteristic for phenoxazinones,whereas no accumulation of 1a was observed for mutantDgriEF (Fig. 2A and B). For experimental convenience in purification andstructural determination of the compounds accumulated inmutant DgriEF, we introduced griR under the control of aforeign promoter on a high-copy-number plasmid pIJ702into mutant DgriEF, since GriR, the pathway-specifictranscriptional activator for the grixazone biosynthesisgenes, activates the transcription of all the grixazonebiosynthesis genes and overexpression of griR leads toenhancement of the yield of grixazones (our unpublisheddata). The plasmid we constructed was pIJ702-griR, inwhich griR under the control of the mel promoter wouldbe expressed in the late exponential and stationary phases.The copy number of pIJ702 is 40 to 300 per chromosome(14). S. griseus DgriEF harboring pIJ702-griR produced2a and 2b in larger amounts during 3 to 5 days cultivation.The amounts of these compounds were about 5-fold aslarge as those produced by mutant DgriEF. Afterpurification of 2a and 2b by HPLC, their structures weredetermined to be 3,4-AHBAL (2a; Fig. 2C) and 3,4-AcAHBAL (2b) by 1H and 13C NMR and high-resolutionelectrospray ionization-time-of-flight mass spectrometry.These findings suggested that 2a or 2b, or both, served as asubstrate of GriF, as we had expected.

Production of GriE and GriF in E. coli – The apo-forms ofMelC2-tyrosinases responsible for melanin production in S.antibioticus (22) and Streptomyces castaneoglobisporus(16), forming a stable complex with MelC1, are usuallyco-purified with MelC1. For production of the Cu-containing, active form of GriF, we constructed pET-griEF,which contained griE under the control of the T7 promoterand griF under the control of the T7lac promoter, and pET-griF containing griF under the control of the T7lacpromoter. We also constructed pET-griE, as a negativecontrol, which contained griE under the control of the T7promoter. The GriF protein encoded by these plasmidswould have the structure of GriF-Leu-Glu-His6. Both theT7 and T7lac promoters in E. coli BL21 (DE3)/pLysSwere inducible by lactose in the medium. Crude lysatesof E. coli harboring pET-griEF and pET-griF showed the

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activity to convert o-aminophenol into 2-aminophenoxazin-3-one (3a, APO; see Fig. 2C),whereas no such activity was detected in a crude lysateprepared from E. coli harboring pET-griE. Thestructural elucidation of APO, which was produced byan in vitro reaction of GriF on o-aminophenol, will bedescribed below. The reaction to yield APO from o-aminophenol is shown in Fig. 3B. The specificactivities of the lysates from E. coli harboring pET-griEF and pET-griF were 0.060 ± 0.003 and 0.0035 ±0.0003 U/mg protein, respectively. We purified GriF from the soluble fraction of E.coli BL21 (DE3)/pLysS harboring pET-griEF byfollowing the activity to convert o-aminophenol intoAPO (3a) by three steps of chromatography (see below).The purified sample, with a specific activity of 63.3 ±6.6 U/mg protein, still contained a small amount ofGriE of 13 kDa, in addition to GriF of 36 kDa, whendetermined by sodium dodecyl sulfate - polyacrylamidegel electrophoresis (SDS-PAGE) (data not shown).GriE contained no histidine-tag. The ratio of GriF andGriE in the final sample, even after Resource PHEcolumn chromatography, was about 10:1. This isperhaps due to the property of GriF to form a complexwith GriE, as is found for MelC2 and MelC1.Consistent with this idea, the activity eluted from a gelfiltration Superdex 200 column at positionscorresponding to 23 kDa (GriF) and 38 kDa (GriF-GriEcomplex). Because of the contamination of GriE in the GriFpreparation, we produced GriF alone in E. coli andpurified by the three steps of chromatography includingCu2+-charged HiTrap Chelating HP affinity columnchromatography, on the assumption that the histidine-tagged GriF would bind to the Cu2+-bound affinitycolumn through the histidine-tag, incorporate Cu2+ intoapo-GriF to form the active form, and elute by 200 mMimidazole, as is observed for the tyrosinase of S.castaneoglobisporus (16). As expected, the step of theHiTrap Cu2+-bound column chromatographyextraordinarily increased the specific activity (Table I).The GriF enzyme thus purified gave a single proteinband of 36 kDa on SDS-PAGE (Fig. 4A). Becausethe GriF preparation showed the specific activity of 65.8± 2.6 U/mg protein, which was almost same as that ofthe purified enzyme from E. coli harboring pET-griEF,we used this sample for further study.

General properties of GriF – We determined generalproperties of GriF by measuring APO formation fromo-aminophenol as a substrate, although 3,4-AHBALwas the best substrate among the various phenols weexamined, as described below. GriF was active over awide pH range of 6.0 - 11.0 with a maximum activity at

pH 8.5 - 10.5 (Fig. 4C(a)) and was most stable at pH 6.5 -8.5 (Fig. 4C(c)). This suggested that alkaline conditionsare suitable for the GriF reaction. The optimaltemperature for GriF at pH 7.0 was 55°C (Fig. 4C(b)).The activation energy was calculated to be 14 kcal/mol onthe basis of the Arrhenius plots of the data. When theenzyme sample was kept at pH 7.0 for 20 min at varioustemperatures, it remained active below 55°C (Fig. 4C(d)). We determined the effects of metals on GriF activity.Our attempts to determine the Cu content using atomicabsorption were hampered by some improperly foldedenzyme and potentially the His-tag. The very faint activityof the lysate prepared from E. coli harboring pET-griF wasascribed to the absence of Cu ions from GriF. Additionof 10 mM Cu2+ ion specifically increased the GriF activityin the lysate by about 20-fold, but Zn2+, Ni2+, Co2+, Mn2+,Fe2+, or Mg2+ exerted almost no effects. Metal-chelatingagents, 10 mM EDTA and o-phenanthroline, hadnegligible effects on the purified GriF sample. Thismeans that the two Cu in the active site of GriF areresistant to these chelating agents, as was found for thetyrosinase from mushroom (25).

Substrate specificity of GriF – As described above, GriFused o-aminophenol as a substrate and yielded APO.The kinetic parameters for various substrates aresummarized in Table II. When o-aminophenols, with theexception of 2-amino-4-methylphenol and 3,4-AHBAL,were used as substrates, the products having aphenoxazinone chromophore were followed by measuringthe absorbance at 433 nm, characteristic forphenoxazinone derivatives. When 2-amino-4-methylphenol was used as a substrate, the quinone imineproduced was dimerized to yield an unknown compoundof 14 carbons, but not a phenoxazinone derivative, whendetermined by 13C NMR analysis. We did not characterizethis compound any further. Production of this compoundwas followed by measuring the absorbance at 433 nm.When 3,4-AHBAL was used as a substrate, thedimerization of the quinone imine produced was very slowand no dimerization product was detected during thereaction period. Production of the quinone imine wasalso followed by measuring the absorbance at 433 nm.The molar extinction coefficienct (e) of the quinone iminewas calculated to be 7,400 by analyzing the products (amixture of the substrate and the product) by HPLC andestimating the amount of the product from the amount ofthe substrate decreased. Similarly, e of the dimersproduced from 3,4-AHBA (3d, APOC), o-aminophenol(3a, APO) and 3-amino-4-methylphenol were calculatedto be 30,000, 9,600, and 7,000, respectively. On the otherhand, we tentatively used the value 30,000 calculated forAPOC for e of the dimer from 4,3-AHBA, because it wasnot efficiently used as a substrate. When catechol was

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used, a yellow product, perhaps o-quinone, wasobtained and its e was calculated to be 2,400. We usedthe value 2,400 also for 3,4-dihydrobenzaldehyde andprotocatechuic acid. Thus, we measured the amountsof the o-quinones from o-diphenol derivatives, thequinone imine from 3,4-AHBAL, and somedimerization products from other o-aminophenols asproducts of the GriF reaction. Although it is notknown why the dimerization of the quinone imine from3,4-AHBAL was very slow, we assume thatdimerization of the quinone imine occurs non-enzymatically, as discussed below. Therefore, tocompare the kinetic parameters of GriF for differentsubstrates, we defined GriF activity on the basis of theamount of the substrate decreased (-d[S]/dt), but not theamount of the product (d[P]/dt). Even though theamounts of the products were measured by using thecalculated or estimated e, the kinetic parameters in TableII do not make serious problems in discussion of thesubstrate specificity of GriF. GriF showed the highest activity toward 3,4-AHBAL to yield the corresponding quinone imine,which in turn dimerized to form 2-aminophenoxazin-3-one-8-aldehyde (APOAL; 3c), as was expected fromthe finding that mutant DgriEF accumulated thiscompound. Formation of APOAL from twomolecules of the quinone imine, in which the –CHOgroup on one of the quinone imine was lost, wasexpected to occur non-enzymatically. We will discussthis reaction later. On the other hand, GriF showedalmost no activity toward 3,4-AcAHBAL, anothercompound accumulated in mutant DgriEF, whichsuggested that the substrate of GriF in the grixazonebiosynthetic pathway was 3,4-AHBAL. 3,4-AcAHBAL is assumed to be a shunt product. Thisidea was supported by the observation that acetylationof the amino group of 3,4-AHBAL and 3,4-AHBAoccurred in S. griseus independently of the geneproducts encoded by the grixazone biosynthesis genecluster (data not shown). GriF also showed highactivity toward 2-amino-4-methylphenol and o-aminophenol. However, 3-amino-4-hydroxybenzoicacid (3,4-AHBA) or 4-amino-3-hydroxyzenzoic acid(4,3-AHBA), both containing a negatively chargedgroup, was not efficiently oxidized. 3-Amino-4-hydroxybenzensulfonic acid was also not used as asubstrate. A vivid contrast between GriF andtyrosinases was that the former showed only slightactivity toward o-diphenol derivatives, such as 3,4-dihydroxybenzaldehyde, catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), and protocatechuicacid. Another contrast between GriF and tyrosinaseswas that GriF showed no monophenolase activity;tyrosine, p-hydroxybenzaldehyde, phenol, o-

nitrophenol, or aniline was not oxidized. GriF did not use some benzoic compounds such asp-hydroxybenzaldehyde, L-tyrosine, 4-hydroxy-3-nitrobenzaldehyde, o-nitrophenol, aniline, phenol, or 3-amino-4-hydroxybenzensulfonic acid. We determinedthe conversion of o-aminophenol into APO in the presenceof these compounds. Of these compounds, p-hydroxybenzaldehyde showed the most potentcompetitive inhibition (Table II and Fig. 4B). 4-Hydroxy-3-nitrobenzaldehyde, containing a nitro group atthe 3 position, also showed significant competitiveinhibition. It was therefore concluded that thesecompounds were bound in the active site of GriF.Interestingly, L-tyrosine, a non-substrate of GriF, showedsignificant competitive inhibition. On the other hand, 3-amino-4-hydroxybenzensulfonic acid showed a very highKi value, suggesting that it cannot be bound strongly in theactive site of GriF.

Hetero-dimerization – As described above, GriF producedAPO when o-aminophenol was used as a substrate. Wedetermined whether heterogeneous dimerization occurswhen catechol is present in the GriF reaction on o-aminophenol. In the presence of o-aminophenol alone,GriF gave a single product of APO (3a; Fig. 5A(a)).However, the co-existence of catechol yielded anadditional product (3b; Fig. 5A(b)), which was identifiedto be 2-hydroxyphenoxazin-3-one (HPO, see Fig. 2C) by1H and 13C NMR and mass spectrometry. The co-existence of catechol in the GriF reactions on 3,4-AHBAL,3-amino-4-methylphenol, 3,4-AHBA, and 4,3-AHBA alsoyielded a probable heterogeneously dimerized product, inaddition to the homogenously dimerized product (data notshown). However, the co-existence of phenol or anilinedid not yield any product other than APO in the GriFreaction on o-aminophenol, when determined byfollowing the absorbance from 210 to 490 nm on HPLC(Fig. 5A(c) and (d)).

In vitro production of grixazone A by GriF – In theoxidation of catechin by tyrosinases and peroxidases in thepresence of –SH compounds, for example, glutathione, theo-quinones produced are non-enzymatically conjugated toglutathione, resulting in formation of catechin-glutathioneconjugates (26). We therefore supposed that in thegrixazone biosynthetic pathway the N-acetylcysteinemoiety would be introduced in the quinone imineproduced from 3,4-AHBAL by GriF by non-enzymaticconjugation via the –SH group. In the reaction of GriFon 5 mM 3,4-AHBAL, peak 4 in Fig. 5B(b) graduallyincreased until 30 min and peak 3c appeared thereafter.The compound in peak 3c was identified to be 2-aminophenoxazin-3-one-8-aldehyde (APOAL; 3c) by 1Hand 13C NMR and mass spectrometry. The compound in

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peak 4 was presumed to be the quinone imine derivedfrom 3,4-AHBAL because its m/z was 135.03334 (M)+

on high resolution electrospray ionization-time-of-flightmass spectrometry (calculated for C7H5N1O2, 1.31mmu error). The quinone imine was detected in theculture broth of S. griseus, which excludes thepossibility that this compound is an artifact produced bythe in vitro reaction. When 1 mM N-acetylcysteinewas present in the GriF reaction on 3,4-AHBAL, a newpeak 1a appeared, in addition to peaks 3c and 4 (Fig.5B(c)). The compound in peak 1a had the sameretention time on HPLC and the same UV-VISspectrum as grixazone A (1a; Fig. 5B(a)). N-Acetylcysteine, a reducing agent, inhibited the GriFreaction and it completely inhibited the reaction at ahigh concentration (5 mM). The addition of N-acetylcysteine occurred before the dimerization to formAPOAL, because no addition of N-acetylcysteine in thereaction mixture containing APOAL instead of 3,4-AHBAL was detected (Fig. 5B(e)). The reaction ofGriF on o-aminophenol in the presence of N-acetylcysteine is illustrated in Fig. 3C.

DISCUSSION

Functions of GriF and GriE – GriF encoded within thegrixazone biosynthesis gene cluster has been found tobe responsible for the formation of the phenoxazinonechromophore of grixazone by oxidative coupling of o-aminophenols. GriF is incapable of oxidation of L-tyrosine, despite that it shows about 50% identity inamino acid sequence to the tyrosinases responsible formelanin production in streptomycetes. Like otherMelC2 tyrosinases in streptomycetes, GriF perhapsaccepts copper ions from GriE, a homologue of MelC1Cu-chaperon. This is the first report to describe theenzyme system responsible for in vivo formation of aphenoxazinone skeleton. Streptomycetes produce avariety of secondary metabolites having aphenoxazinone chromophore, and some of thetyrosinase homologues are supposed to be responsiblefor the formation of phenoxazinones. Since thebiosynthesis genes for a given secondary metabolite arealways organized as a gene cluster, we can predict that atyrosinase homologue encoded within the gene clusterfor a secondary metabolite containing a phenoxazinonechromophore is involved in the formation of thephenoxazinone. We can safely say that thephenoxazinone chromophores of michigazone, 4-demethoxymichigazone, exfoliazone, and texazone, allhaving a functional group at the 8-position, are formedfrom o-aminophenol derivatives by the action of GriFhomologues.

GriF as a novel o-aminophenol oxidase – Tyrosinase is amember of the type-3 copper protein family, to whichhemocyanins and catechol oxidases also belong. On thebasis of the catalytic properties of tyrosinases and the X-ray crystallography of the binuclear copper active sites ofhemocyanin (27) and catechol oxidase (28), detailedmechanisms of tyrosinase reaction as both amonophenolase and a diphenolase have been proposed (29,30). Some tyrosinases seem to have generous substratespecificity; the tyrosinases from N. crassa (12), mushroom(13), and S. glaucescens (12) catalyze the oxidation ofaromatic amines, o-diamines, and o-aminophenols, but at alow rate, in addition to phenol and catechol. The presentstudy has revealed a unique substrate specificity of GriF,which we can classify into an o-aminophenol oxidase.First, GriF prefers o-aminophenol as a substrate to catechol,whereas tyrosinases prefer catechol-type substrates (o-diphenols) to o-aminophenols. The kcat/Km value for 3,4-dihydroxybenzaldehyde is 25 times smaller than that for3,4-AHBAL, due solely to the decrease in the kcat value for3,4-dihydroxybenzaldehyde. Concerning very lowoxidation rates of aromatic amines and o-aminophenols bytyrosinases, Gasowska et al. (13) pointed out that o-diphenols are deprotonated more readily than arylaminesdue to a large difference in the pKa value between phenols(pKa = 10) and arylamines (pKa = 27). In addition, GriFhad a rather small kcat value (14.0 ± 1.1 s-1) for the bestsubstrate, 3,4-AHBAL, when compared with that (390 ±16 s-1) of the tyrosinase for catechol. We thereforeassume that GriF shows high specificity for o-aminophenols by decreasing the catalytic activity on o-diphenols. Second, GriF shows no monophenolase activity,whereas tyrosinases show monophenolase activity. LikeGriF, catechol oxidase shows diphenolase activity but notmonophenolase activity (28, 31, 32). On the basis of aparamagnetic study of the interaction between amonophenolic inhibitor p-nitrophenol and the tyrosinasefrom S. antibioticus, together with the X-raycrystallography of a plant catechol oxidase, Tepper et al.(32) proposed that the phenyl ring of Phe-261 of catecholoxidases prevents the ortho protons of the substratephenolic ring from its direct approach toward the catalyticcenter. In fact, an amino acid alignment of variouscatechol oxidases and tyrosinases shows that a Pheanalogous to Phe-261 of catechol oxidases is absent intyrosinases. Interestingly, the amino acid corresponding toPhe-261 in GriF is Tyr, which also has a phenyl ring as aside chain (Fig. 1B). The absence of monophenolaseactivity in GriF may be ascribed to the blocking activity ofthis Tyr residue. Third, GriF activity to oxidize o-aminophenolsdepends on the functional group at the para position to thephenolic hydroxyl group. The kcat/Km value for 2-amino-

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4-methylphenol, containing a –CH3 group at thisposition, was similar to that for 3,4-AHBAL, containinga –CHO group, whereas that for o-aminophenolcontaining no functional group at this position wasdecreased by 4.1 times, mainly due to an increase in theKm value. Furthermore, the kcat/Km value for 3,4-AHBA containing a –COOH group at this position wasdecreased by 130 times compared to that for 3,4-AHBAL. GriF showed no activity on 3-amino-4-hydroxybenzensulfonic acid containing a –SO3H groupat this position. The kinetic parameters for diphenolsshowed a similar tendency to those for o-aminophenols;the kcat/Km value for 3,4-dihydroxybenzaldehydecontaining a –CHO group at the corresponding positionwas 3 and 150 times larger than that for catecholcontaining no functional group at this position and thatfor protocatechuic acid containing a –COOH group,respectively. Therefore, the functional group at thepara position with respect to the hydroxyl group of o-aminophenol is important in binding to GriF and a–CHO group at this position is optimum for the GriFreaction. GriF appears to dislike the compoundscontaining a negatively charged group at this position.X-ray crystallography of GriF, as well as a tyrosinase,will give light on these unique properties in substratespecificity and catalysis of GriF.

A putative model for the coupling of o-aminophenols –The phenoxazinone synthase, an oligomericmulticopper oxidase, from S. antibioticus catalyzes theoxidative condensation of two molecules of 4-methyl-3-hydroxyanthranilic acid to actinocin in vitro, thephenoxazinone chromophore of actinomycin D (33).A mechanism for the reaction was studied by means ofblocking the reaction at various stages with substitutedo-aminophenols (33). According to this mechanism,o-aminophenols are converted to the highlyelectrophilic quinone imines by a two-electronoxidation, which then conjugate to another molecule ofthe o-aminophenols in the catalytic site of the enzyme.The coupling is initiated by a nucleophilic attack on thecarbon at the para position with respect to the ketonegroup of the quinone imine by the amino group of theo-aminophenol. The conjugates are further oxidizednon-enzymatically by two electrons to yield the p-quinone imines. The second intramolecularconjugation of the p-quinone imine, followed by a finaltwo-electron oxidation to give the phenoxazinonechromophore, occurs non-enzymatically out of theactive site. On the other hand, Simandi et al. (34)analyzed the kinetics and mechanism of theferroxime(II)-catalyzed biomimeric oxidation of o-aminophenol by dioxygen as a functionalphenoxazinone synthase model. Because a 4-

substituted 2-aminophenoxyl free radical was detected bythe ESR technique as a reaction intermediate, theyproposed a model in which two molecules of the quinoneimine produced from o-aminophenol were coupled. Doquinone imines react with o-aminophenols or anothermolecule of quinone imines in the formation ofphenoxazinones through the oxidation of o-aminophenolsby GriF? If the coupling of o-aminophenol by GriF isinitiated by the nucleophilic attack on the carbon at thepara position with respect to the ketone group of thequinone imine by the amino group of the o-aminophenol,the presence of aniline in the GriF reaction on o-aminophenol would yield an aniline-quinone imineconjugate (Fig. 3B). If the coupling is initiated by theelectrophilic attack on the carbon at the para position withrespect to the hydroxy group of o-aminophenol by theimino group of the quinone imine, the presence of phenolin the GriF reaction on o-aminophenol would yieldphenoxazin-3-one and/or its reaction intermediate (Fig.3B). However, no such conjugates were produced. Onthe other hand, the presence of catechol, a substrate of GriF,resulted in the formation of a conjugate, HPO (3b, Fig. 3B).We therefore speculate that the coupling of two moleculesof o-aminophenol in the GriF reaction occurs between twomolecules of the quinone imine produced (Fig. 3A).GriF catalyzes only the oxidation of o-aminophenols toyield the corresponding quinone imines and the couplingperhaps occurs non-enzymatically out of the active site,unlike the phenoxazinone synthase. Consistent with thisidea, when 3,4-AHBAL was used as a substrate, anoverwhelmingly larger number of the quinone iminemolecules than that of the GriF molecules was producedduring an early stage of the reaction and then thephenoxazinone began to be produced (Fig. 5B). The oxidation of 3,4-AHBAL and 3,4-AHBA byGriF gave APOAL and APOC, respectively. In thisreaction, the –CHO or –COOH group of one of the twosubstrate molecules was lost. A similar observation wasreported by Hughes et al. (35); 4,3-AHBA in a minimalmedium was oxidized non-enzymatically to yield 2-aminophenoxazin-3-one-7-carboxylate with a loss of a–COOH group. We also observed the formation ofAPOC when 10 mM 3,4-AHBA in 50 mM sodiumphosphate buffer (pH 7.0) was shaken vigorously at 30˚Cfor 12 h (data not shown), showing that the formation ofAPOC from 3,4-AHBA, accompanied by a loss of the–COOH group of one of the two 3,4-AHBA molecules,can occur non-enzymatically. The formation of APOALfrom 3,4-AHBAL is also presumed to occur non-enzymatically, although the reaction is much slower.

The role of GriF in the grixazone biosynthesis – S. griseusmutant DgriEF accumulates 3,4-AHBAL, which is thesubstrate of GriF. We have identified GriCD to be

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responsible for reduction of 3,4-AHBA to yield 3,4-AHBAL (our unpublished data). These findingssupport the prediction of Gould et al. (36) that thephenoxazinones of michigazone, exfoliazone, and 4-demethoxymichigazone might be synthesized from 3,4-AHBA. Our in vitro synthesis of grixazone A from N-acetylcysteine and 3,4-AHBAL in the presence of GriF(Fig. 5B(c)) gives us a hint to establish the biosyntheticpathway of grixazone (Fig. 3C), on the basis of theorganization of the grixazone biosynthesis genes.Because grixazone A is not formed from N-acetylcysteine and APOAL in the presence of GriF (Fig.5B(e)), the addition of N-acetylcysteine to the quinoneimine derived from oxidation of 3,4-AHBAL by twoelectrons occurs before its dimerization with anothermolecule of the quinone imine. In the oxidation ofcatechin by tyrosinases and peroxidases in the presenceof –SH compounds, for example, glutathione, the o-quinones produced are non-enzymatically conjugated toglutathione, resulting in the formation of catechin-glutathione conjugates (26). We therefore assume thatin the grixazone biosynthetic pathway the quinoneimine produced from 3,4-AHBAL by GriF is non-enzymatically conjugated by the –SH group of N-acetylcysteine and then is dimerized with anotherquinone imine, resulting in formation of grixazone A.The non-enzymatic conjugation of N-acetylcysteine tothe quinone imine is consistent with the fact that withinthe grixazone biosynthesis gene cluster there is noplausible gene encoding an enzyme able to attach N-acetylcysteine to the phenoxazinone skeleton. Grixazone B (1b, Fig. 2C) was not formed fromN-acetylcysteine and 3,4-AHBA in the presence of GriF，although APOC (3d) was formed (data not shown).The coupling of the quinone imines produced from 3,4-AHBA by the action of GriF is rather rapid, althoughthat of the quinone imines from 3,4-AHBAL is slow.This may facilitate the introduction of N-acetylcysteineto the quinone imine from 3,4-AHBAL. Therefore,the conversion from 3,4-AHBA to 3,4-AHBAL isimportant in the grixazone biosynthesis. The substratespecificity of GriF, unfavorable to the compoundshaving a negatively charged substituent at the paraposition with respect to the hydroxyl group of o-aminophenols, supposedly restrain the formation of ashunt product, APOC, as a result of oxidation of 3,4-AHBA by GriF, in the biosynthesis of grixazone.

ACKNOWLEDGMENTS

We should like to thank Shohei Sakuda for advicein the structure analysis of 3,4-AHBAL. This workwas supported by a grant from the Industrial TechnologyResearch Grant Program in 2003 of the New Energy and

Industrial Technology Development Organization of Japan(03A07002), by a Grant-in-Aid for Scientific Research onPriority Areas from Monkasho, and by the BioDesignProgram of the Ministry of Agriculture, Forestry, andFisheries of Japan.

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38. Wichers, H. J., Recourt, K., Hendriks, M., Ebbelaar, C. E., Biancone, G., Hoeberichts, F. A.,Mooibroek, H., and Soler-Rivas, C. (2003) Appl. Microbiol. Biotechnol. 61, 336-341

39. Kwon, B. S., Haq, A. K., Pomerantz, S. H., and Halaban, R. (1987) Proc. Natl. Acad. Sci. USA 84,7473-7477

FOOTNOTES

The nucleotide sequence of the griEF locus has been submitted to the DDBJ / GenBankTM / EBI Data Bankwith accession number AB214954. 1 The abbreviations used are: 3,4-AcAHBAL; 3-acetylamino-4-hydroxybenzaldehyde; 3,4-AHBA, 3-amino-4-hydroxybenzoic acid; 4,3-AHBA, 4-amino-3-hydroxybenzoic acid; 3,4-AHBAL, 3-amino-4-hydroxybenzaldehyde; APO, 2-aminophenoxazin-3-one; APOAL, 2-aminophenoxazin-3-one-8-aldehyde; APOC,2-aminophenoxazin-3-one-8-carboxylate: DMSO, dimethylsulfoxide; L-DOPA, L-3,4-dihydroxyphenylalanine;FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography; HPO, 2-hydroxyphenoxazin-3-one; PHS, phenoxazinone synthase; SDS-PAGE, sodium dodecyl sulfate - polyacrylamidegel electrophoresis; TAT, twin-arginine translocation.

FIGURE LEGENDS

FIG. 1. Alignment of amino acid sequences covering the two copper-binding sites (CuA-site and CuB-site) ofGriF and tyrosinase family members (A) and the organization of the grixazone biosynthesis gene cluster (B). A,The three histidine residues (▼) responsible for copper binding by each Cu-binding site and the position (●)corresponding to Phe-261 of catechol oxidase I in Ipomoea batatas (IbCO; GenBankTM accession number,Q9ZP19 (28)) are indicated. The tyrosinases aligned are those from S. antibioticus (SaMelC2; B23971 (17)); S.castaneoglobisporus (ScMelC2; AAP33665 (20)); S. glaucescens (SgMelC2; A24089 (18)); S. lavendulae(SlMelC2; 2113331B (21)); N. crassa (NcTYR; YRNC (37)); mushroom (Agaricus bisporus; AbTYR;CAA59432 (38)); and Homo sapiens (HsTYR; NP_000363 (39)). B, The 1.1 kb region covering the griE-griFregion was deleted by two times of homologous recombination. Correct deletion was checked by Southernhybridization with the 0.5 kb fragment as 32P-labeled probe.

FIG. 2. HPLC chromatograms of the culture broths prepared from the wild-type strain, S. griseus IFO13350, andmutant DgriEF (A and B) and the chemical structures of grixazones and related compounds (C). A, The culturebroth of the wild-type strain after 3 days of cultivation at 26.5°C in SMM medium was analyzed by measuring theabsorbance at 254 nm. In the inset, the HPLC profile for the culture broth after 5 days, measured by absorbance at433 nm, is shown. B, The culture broth of mutant DgriEF after 3 days of cultivation at 26.5°C in SMM mediumwas analyzed by measuring the absorbance at 254 nm. In the inset, the HPLC profile for the culture broth after 5days, measured by absorbance at 433 nm, is shown. AUFS, absorbance unit full scale. C, The structures ofgrixazones, 3,4-AHBAL, 3,4-AcAHBAL, and related compounds are shown.

FIG. 3. Catalytic properties of GriF. A, A putative pathway of coupling o-aminophenols. GriF catalyzes theoxidation of o-aminophenols to yield the corresponding quinone imines, which then non-enzymatically coupleswith another molecule of the quinone imine. B, GriF yields APO (3a) from o-aminophenol, as a result ofhomogeneous coupling. Co-existence of catechol in the GriF reaction on o-aminophenol results in the formationof HPO (3b) as a result of heterogenous coupling, in addition to APO (3a). C, In vitro synthesis of grixazone A(1a) by GriF in the presence of 3,4-AHBAL (2a) as a substrate and N-acetylcysteine (NAC) as a –SH group-containing compound.

FIG. 4. Properties of GriF. A, SDS-polyacrylamide gel electrophoresis of the GriF sample purified from E. coliBL21 (DE3)/pLysS harboring pET-griF. Myosin (200 kDa), b-galactosidase (116 kDa), bovine serum albumin(67 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soy bean trypsin inhibitor (21 kDa), lysozyme (14kDa), and bovine aprotinin (6 kDa) were used for the molecular mass standards. B, Competitive inhibition of theGriF reaction on o-aminophenol by p-hydroxybenzaldehyde. The concentrations of the inhibitor were 0 mM (●),0.5 mM (■), 1.0 mM (▲), 2.5 mM (○), 5.0 mM (□), and 10 mM (△). C, Enzymatic properties of GriF.

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Standard reaction mixtures containing 0.0043 U of GriF were incubated at 30°C for 7 min at various pHs (a) and atvarious temperatures at pH 7.0 for 7 min (b), and the amounts of the APO formed were measured. The buffersused in (a) and (c) were 50 mM sodium phosphate-50 mM sodium citrate (pH range, 4 to 11) and 50 mM sodiumphosphate, respectively. After GriF had been kept at 60°C for 20 min at various pHs, the GriF activity wasassayed at pH 7.0, 30˚C for 7 min by measuring the amounts of the APO formed (c). After GriF had been kept atpH 7.0 for 20 min at various temperatures, the GriF activity was measured at pH 7.0, 30˚C for 7 min (d).

FIG. 5. HPLC analysis of the products formed by the action of GriF on various substrates. AUFS, absorbanceunit full scale. A, Heterogenous coupling between the quinone imine and catechol. Standard reaction mixturescontaining 5 mM each of the substrates were incubated at 30°C for 1 h and applied to HPLC. The reactionmixtures contained o-aminophenol alone (a), o-aminophenol and catechol (b), o-aminophenol and phenol (c), ando-aminophenol and aniline (d). Co-existence of catechol yielded HPO (3b), in addition to APO (3a). B, In vitrosysnthesis of grixazone A by GriF from 3,4-AHBAL and N-acetylcysteine. The standard reaction mixturescontaining 5 mM 3,4-AHBAL and 1 mM N-acetylcysteine were incubated at 30°C for 1 h and applied to HPLC.(a) The authentic sample of grixazone A. (b) GriF yielded peak 4, containing the quinone imine, in addition toAPOAL (3c). (c) The presence of 3,4-AHBAL and N-acetylcysteine resulted in the formation of grixazone A(1a), in addition to the quinone imine (4) and APOAL (3c). (d) The reaction mixture without GriF, as a negativecontrol, yielded no products. (e) The reaction mixture containing 1 mM N-acetylcysteine and 0.1 mM APOAL(3c) did not yield grixazone A (1a).

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a Because apo-GriF bound Cu2+ and became active during the HiTrap Chelating Cu2+ column chromatography, thespecific activity was dramatically increased after this step.

Purification step Total protein(mg)

Specific activitya

(U/mg protein)Yield(%)

Purification(-fold)

Crude extract 64.00 00.0035 0,100 000001

HiTrap Chelating Cu2+ 01.30 11.0000 6,400 03,100

HiTrap Q 00.42 17.0000 3,300 05,000

Resource PHE FPLC 00.16 66.0000 4,700 19,000

Table I

Purification of recombinant GriF

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Relative kcat / Km (%) Substrate kcat (s-1) Km (mM) kcat / Km (s-1 M-1)

GriF Tyrosinase Ki (mM)

o-Aminophenol derivatives o -Aminophenol .0020 ± 3.000 03.5 ± 0.60 .05,800 ± 170,0. < 100.00 < 0,160.00 3,4-AHBAL .0014 ± 1.000 0.58 ± 0.08 .24,000 ± 1,400. < 420.00 < 0,000.10 3,4-AcAHBAL 00< 0.01 0,00< 0.01 2-Amino-4-methylphenol .0018 ± 1.000 0.75 ± 0.06 .23,000 ± 1,200. < 400.00 < 0,089.00 3,4-AHBA N.D. N.D. .00,190 ± 5,000. < 003.20 < 0,000.04 4,3-AHBA N.D. N.D. 0,007.3 ± 0.100, < 000.13 < 0,000.08 3-Amino-4-hydroxybenzensulfonic acid 00< 0.01 < 0,000.01 .031 ± 7.0

o-Diphenol derivatives 3,4-Dihydroxybenzaldehyde 00.80 ± 0.090 0.41 ± 0.06 .00,970 ± 40,00. < 017.00 < 0,000.21 Catechol .0012 ± 1.000 .019 ± 2.00 .00,320 ± 3,000. < 005.60 < 2,500.00 Protocatechuic acid N.D. N.D. 00,06.3 ± 0.3,00 < 000.11 < 0,005.90 L-DOPA 0.066 ± 0.016 05.5 ± 1.80 .00,012 ± 1,000. < 000.21 < 0,100.00

Other compounds L-Tyrosine 00< 0.01 < 0,082.00 03.2 ± 0.6 p-Hydroxybenzaldehyde 00< 0.01 0,00< 0.01 01.9 ± 0.2 4-Hydroxy-3-nitrobenzaldehyde 00< 0.01 0,00< 0.01 03.9 ± 0.3 o-Nitrophenol 00< 0.01 0,00< 0.01 06.1 ± 0.6 Aniline 00< 0.01 0,00< 0.01 .014 ± 1.0 Phenol 00< 0.01 < 0,012.00 .021 ± 3.0

Table II Kinetic parameters for the GriF activity

N.D., the Km value was too large to be determined.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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HorinouchiHirokazu Suzuki, Yasuhide Furusho, Tatsuichiro Higashi, Yasuo Ohnishi and Sueharu

chromophore of grixazoneA novel ortho-aminophenol oxidase responsible for formation of phenoxazinone

published online November 10, 2005J. Biol. Chem. 

  10.1074/jbc.M505806200Access the most updated version of this article at doi:

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