Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)-geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (؉)geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.
Phenol oxidative coupling is one of the most important reactions in the biosynthesis of natural products. In 1957, Barton and Cohen (1) proposed that new C-C or C-O bonds of either intra-or intermolecules could be formed by the pairing of radicals that are produced by one-electron oxidation of phenols. Since then, importance of phenol oxidative coupling reaction that is involved in the biosynthesis of a wide range of natural products such as alkaloids (e.g. morphine, lycorine), lignans, and other phenolic compounds (e.g. usunic acid, griseofulvin) of versatile origins has been recognized (2). Therefore, a variety of chemical and biological oxidizing systems has been developed for oxidations of phenolic compounds (3)(4)(5). However, model enzyme systems using laccase, tyrosinase, and peroxidase have been unable to reproduce regio-and stereospecificities, which are often observed in natural product biosynthesis. Hence, elucidation of the true nature of the biocatalysts involved in specific biosynthetic phenol oxidative couplings has been the subject of intensive research for many years.
Fungi produce a group of compounds called grisans. Formation of the unique spiro structures of grisans such as griseofulvin, geodin, and their derivatives from corresponding benzophenone precursors had been thought to be catalyzed by the specific phenol oxidative coupling enzymes (6). Enzymatic formation of grisan structure of geodin was first demonstrated by Komatsu (7) using cell-free extracts of Penicillium estinogenum. The enzyme, which was suggested to be copper protein, catalyzed regio-and stereospecific formation of (ϩ)-geodin from benzophenone dihydrogeodin. On the other hand, model enzyme systems using laccase or peroxidase gave optically inactive geodin (8). No specific enzyme activity has been detected for dehydrogriseofulvin formation.
We have been working on the biosynthesis of (ϩ)-geodin in Aspergillus terreus (Fig. 1) at the enzyme level (9 -13) and previously reported the purification of dihydrogeodin oxidase (DHGO) 1 from A. terreus (9), which catalyzes the regio-and stereospecific formation of (ϩ)-geodin from dihydrogeodin. This was the first example of purification and characterization of the specific phenol oxidative coupling enzyme. The purified DHGO showed an intense blue color and had absorption maximum at 600 nm. The EPR spectrum of DHGO clearly indicated the presence of type-1 and type-2 copper atoms in the enzyme molecule. These facts suggested that DHGO is a multicopper blue protein. Similar properties were also reported for sulochrin oxidases from Penicillium frequentans and Oospora sulfrea-ochracea by Nordlöv and Gatenbeck (14). Both enzymes catalyze specific formation of bisdechlorogeodin but in opposite stereospecificity. Recently, we purified sulochrin oxidase from P. frequentans, 2 although the clarified properties were found to be quite different from those reported by Nordlöv and Gatenbeck.
Zenk et al. (15) reported that cytochrome P-450 enzymes catalyze phenol oxidative coupling reactions involved in benzylisoquinoline alkaloid biosynthesis. Recently, they purified a cytochrome P-450 enzyme that mediates specific intermolecular phenol oxidative coupling reaction to form bisbenzylisoquinoline alkaloid berbamunine (16). Presence of peroxidaselike stereoselective phenol coupling enzyme is reported for lignan biosynthesis (17). Therefore, it is very interesting to know how the same type of phenol oxidative coupling reactions are driven by the different kind of oxidation enzymes such as cytochrome P-450s, peroxidase-like enzymes in plants, and * This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) D49538.
To elucidate the precise nature of the biocatalysts involved in the specific phenol oxidative coupling reactions, we decided to clone the gene for DHGO from A. terreus. In this paper, we describe the cloning, sequencing, and heterologous expression of the DHGO gene as the first example of a multicopper blue protein that catalyzes regio-and stereospecific phenol oxidative coupling reaction.
A. terreus strain IMI 16043 was used, and its culture condition was previously described (9). Aspergillus nidulans strain FGSC 89 (argB, biA) was maintained on arginine and biotin-supplemented Aspergillus minimal medium described by Pontecorvo et al. (19). A. nidulans strain FGSC 4 (wild type) was obtained from Fungal Genetics Stock Center (The University of Kansas Medical Center, Kansas). P. frequentans CMI 96659 was obtained from Commonwealth Mycological Institute (United Kingdom), which was maintained and cultured in the same way as that of A. terreus IMI 16043 (9).
Vectors-The expression phage vector gt11 was obtained from Promega. ZAP II and pBluescripts were from Stratagene. pT7 Blue from Novagen was used for direct cloning of polymerase chain reaction products. pTAex3 3 is a shuttle vector carrying the ampicillin resistance gene and the argB gene of A. nidulans for selection in E. coli and arginine prototrophy selection in fungi, respectively. The plasmid also carries the promoter and terminator region of the Taka-amylase A gene (amyB) of Aspergillus oryzae (20) flanking unique EcoRI and SmaI cloning sites. pDH25 was a generous gift from Cullen et al. (21). It contains the E. coli hygromycin B phosphotransferase gene fused to the promoter of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase gene and the termination region of A. nidulans trpC gene.
Purification of Dihydrogeodin Oxidase from A. terreus-The native DHGO was purified from A. terreus as previously described (9) with the following modification. After hydroxyapatite column, the sample was purified by Mono Q HR 5/5 anion exchange column (Pharmacia Biotech Inc.) equilibrated with 0.025 M Tris-HCl buffer (pH 7.0). The enzyme was eluted with a linear gradient of NaCl concentration from 0 to 1 M. Then, active fractions were applied to a chromatofocusing column Mono P HR 5/5 (Pharmacia) equilibrated with Bistris-HCl buffer (pH 6.3). Elution was carried out by polybuffer 74, pH gradient from 7.4 to 4.0, with co-gradient of NaCl from 0 to 0.5 M. The eluted enzyme was further purified by HPLC gel filtration on TSK G-3000 SW column (7.5 ϫ 600 mm, Tosoh, Japan) with 0.2 M potassium phosphate buffer (pH 6.0) containing 0.2 M NaCl.
The N-terminal amino acid sequence of the purified DHGO was determined as described (22) using Applied Biosystems 473A peptide sequencer according to the manufacturer's instructions. Antibody Production-Polyclonal antibodies recognizing DHGO were obtained by immunizing rabbits with the purified native DHGO first with complete Freund adjuvant and then with incomplete Freund adjuvant as a booster 2 weeks after first immunization. IgG fraction was obtained by passage through a protein A affinity column.
SDS-PAGE and Western Blot Analysis-SDS-PAGE was performed essentially as described by Laemmli (23). Gels of 10.5% acrylamide and 0.5% bisacrylamide were used and electrophoresed in a modified Laemmli buffer system (0.025 M Tris base, 0.192 M glycine, 0.05% SDS). Gels were stained with a silver stain kit of Kanto Chemicals (Japan) or Coomassie Brilliant Blue using Quick CBB kit (Wako Pure Chemicals, Japan). After separation on SDS-PAGE, proteins were blotted onto nitrocellulose membrane (Schleicher and Schuell) by Bio-Rad electroblotting apparatus according to the manufacturer's instructions. The filter was detected by enzyme-linked immunodetection with anti-DHGO antibody and anti-rabbit IgG-peroxidase conjugate using Konica immunostain horseradish peroxidase kit (Konica, Japan).
Fungal Genomic DNA Isolation and Southern Blot Analysis-Genomic DNAs of fungi were isolated by the modified procedure of Biel and Parrish (24) previously described (25).
Construction and Screening of cDNA Libraries-The first cDNA library was constructed from A. terreus mRNA in the expression phage vector gt11 using cDNA synthesis kit (Boehringer Mannheim) according to the manufacturer's instruction. A library containing 3.5 ϫ 10 5 recombinants was made, and its 1.4 ϫ 10 5 plaques were screened.
Screening of the first cDNA expression library was carried out as follows. After plaque lift on nitrocellulose filter, plaques presenting DHGO epitopes by isopropyl-1-thio-␤-D-galactopyranoside induction were detected by anti-DHGO primary antibody and anti-rabbit IgG peroxidase conjugate secondary antibody using Konica Immunostain horseradish peroxidase kit.
The second cDNA library was screened by plaque hybridization using a 280-bp cDNA fragment cloned from the first cDNA library as a probe. Labeling of the probe DNA with digoxigenin-dUTP was done by DIG DNA labeling kit (Boehringer Mannheim). Hybridization was carried out in a solution containing 5 ϫ SSC, 0.1% SDS, 1% blocking reagent solution, 0.02% sodium N-lauroyl sarcosine at 60°C overnight. Filters were washed twice with 0.1 ϫ SSC, 0.1% SDS at 60°C for 30 min and then detected by DIG nucleic acid detection kit (Boehringer Mannheim) using nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate.
Selected ZAP II recombinant phages were rescued to pBluescript recombinants by helper phage superinfection as described in the ZAP II manufacturer's manual (Stratagene).
Construction and Screening of A. terreus Partial Genomic DNA Library-A. terreus genomic DNA was digested with restriction endonuclease SacI, and the digestion products were separated by agarose gel electrophoresis. DNA fragments of approximately 5-7 kb recovered by Suprec-01 filtration (Takara Shuzo, Japan) were ligated with SacI cleaved and dephosphorylated ZAP II phage DNA arms. Packaging of the ligation mixture gave a library comprising of 1.4 ϫ 10 6 plaqueforming units.
Screening of the genomic DNA library (5 ϫ 10 4 plaques) was carried out in the same way as that of second cDNA library.
A rapid amplification of cDNA ends method (28) was used to extend the 5Ј-end of DHGO cDNA. One microgram of poly(A) ϩ RNA was reverse transcribed using P3 primer. The reverse-transcribed first strand cDNA was polyadenylated by terminal deoxynucleotidyltransferase (Toyobo, Japan). P2 primer and XbaI-dT 15 primer adaptor were used to perform amplification at 94°C for 1 min, at 50°C for 1 min, and 72°C for 1 min for 30 cycles. The cDNA fragment was then directly subcloned into pT7 Blue. The result was confirmed by DNA sequencing.
DNA Sequence Analysis-DNA sequence analysis was carried out by dideoxy chain termination method using Sequenase version 2.0 (Amersham) and fluorescein isothiocyanate-labeled primer or terminator (Yuki Gosei Kogyo Co. Ltd., Japan) with Hitachi auto DNA sequencer SQ-3000. Fragments for sequencing were generated either by restriction digestion or deletion with exonuclease III and mungbean nuclease. DNA sequence data were organized and analyzed using the DNASIS program (Hitachi Software Engineering Co., Ltd., Japan) and DNA analysis package of Human Genome Center (Institute of Medical Science, The University of Tokyo). The final sequence of the cloned genomic DNA and cDNA for dihydrogeodin oxidase was determined from both strands.
Fungal Transformation-Protoplasts of A. nidulans and P. frequentans were prepared by the method of Tilburn et al. (29) and Huang et al. (25). Transformation was according to the procedures described by Gomi et al. (30). Stable transformants were isolated by repeated transfer of sporulating colonies on selection plates.
Enzyme Assay and HPLC Conditions-DHGO activity was assayed as previously described (9). HPLC analysis of DHGO reaction product was performed on ODS-80 TM column (Tosoh) using CH 3

RESULTS
Isolation of cDNA for Dihydrogeodin Oxidase from A. terreus-The first A. terreus cDNA library constructed in gt11 was screened with polyclonal antibody against DHGO. Four positive phage clones were obtained from 1.4 ϫ 10 5 recombinant phages (DHGO21, DHGO22, DHGO41, DHGO81). Sequencing analysis showed that DHGO21 and DHGO22 were the same clones containing 280-bp inserts. DHGO41 and DHGO81 contained shorter inserts with deletion before the poly(A) tail. A data base homology search indicated some homology with oxidation-related enzyme genes. The quality problem of the first cDNA library, e.g. insert sizes, led us to construct the second cDNA library of A. terreus. By screening the new cDNA library by plaque hybridization, four positive clones were obtained from 4 ϫ 10 4 recombinant phages. These four clones were in vivo rescued to pBluescript recombinants and found to contain EcoRI fragments of 1 and 1.6 kb (cDNA 1ϳ4 in Fig. 2). Sequence analysis showed that they are all derived from the same mRNA species. However, Northern blot analysis indicated that the size of mRNA for DHGO is about 2.5 kb. Also, subunit size of DHGO (76 kDa) suggested that mRNA size is larger than 2 kb. To clone the 5Ј-end of DHGO cDNA, rapid amplification of cDNA ends method was applied. The template in this reaction was cDNA synthesized from mRNA of 3-day-old A. terreus mycelium using P3 primer and then polyadenylated. A DNA fragment comprising the 5Ј-end of the cDNA amplified using P2 primer and XbaI-dT 15 primer adaptor was cloned into pT7 Blue and sequenced (cDNA 5 in Fig. 2).
Isolation of the Genomic DNA Encoding Dihydrogeodin Oxidase-To isolate the genomic DNA clones encoding DHGO, genomic Southern analysis was performed using the 280-bp cDNA fragment obtained from the first cDNA library as a probe. Southern blot analysis of BamHI-digested A. terreus genomic DNA gave two hybridizing bands of about 15 kb and 800 bp since there is a single BamHI site in the 3Ј-non-coding region of the cloned 280-bp cDNA. cDNA coding region probe indicated that the 800-bp band is the 5Ј-upstream region of DHGO gene. To know the suitable restriction enzyme fragment for genomic DNA cloning, single digestion of A. terreus genomic DNA with variable restriction enzymes or double digestion together with BamHI were carried out. By Southern blot analysis, a 5.5-kb band by SacI digestion and 800-bp band by SacI and BamHI digestion were detected with the cDNA probe. This result indicated that 1) the 5.5-kb SacI fragment contained a 5Ј-coding region including the BamHI 800-bp fragment and 2) the distance of 3Ј-downstream SacI site from the BamHI site in the 3Ј-non-coding region is very short because no detectable band other than 800 bp was observed in double digestion analysis, suggesting that DHGO is most likely encoded by a single copy gene, and the whole coding region of DHGO gene exists in the 5.5-kb SacI fragment.
A partial genomic DNA library of size-fractionated SacIdigested fragments around 5 kb was constructed using ZAP II phage vector and screened with the DIG-labeled 280-bp cDNA probe. Five positive clones selected were in vivo rescued to pBluescript recombinants, and all contained the 5.5-kb SacI fragments (pBSDHGO). The restriction endonuclease map of this 5.5-kb SacI fragment and the DHGO coding region located in the 2.8-kb BamHI fragment are illustrated in Fig. 2.
Structural Organization of the Gene for Dihydrogeodin Oxidase-The nucleotide sequence of the region corresponding to the DHGO genomic DNA was determined as shown in Fig. 3. Sequence analysis of the 5Ј-flanking region revealed the presence of structural features considered to be important for gene transcription. A TATA box-like sequence (TATAAA) was found 214 bp upstream from the presumptive translation start (ATGCCG), and a CAAT element (CAAAT) was located at 437 bp upstream from the ATG.
A canonical consensus sequence for the polyadenylation signal of higher eukaryotes, AATAAA (32), is not present in the C-terminal of the DHGO gene. The indicated methionine starts an open reading frame comprised of 605 amino acids, and the N-terminal amino acid of the purified enzyme is found in the same open reading frame after the 21-amino acid leader sequence, as shown in Fig. 3. The molecular mass of the mature DHGO deduced from the amino acid sequence was 68 kDa, whereas that estimated by SDS-PAGE was 76 kDa (9). Since the predicted DHGO peptide carried six potential N-glycosylation sites (Asn-X-Ser or Thr) (33) at amino acids 27, 106, 111, 282, 467, and 483, post-translational modification by glycos-ylation might have occurred. The fact that digestion of the purified native enzyme with N-glycanase (34) increased the mobility on SDS-PAGE (Fig. 4) revealed the existence of Nglycosylation in the mature DHGO protein.
Sequence Comparison of Dihydrogeodin Oxidase with Other Blue Copper Proteins-The deduced amino acid sequence of the DHGO from A. terreus was compared with other multicopper blue proteins to analyze the structural relationships among them.
Closer inspection of the sequences revealed four conserved regions (A-D) shown in Fig. 5. These highly homologous regions contain clusters of histidine residues, which constitute the proposed 12 copper binding ligands to type-1, type-2, and type-3 coppers, on the basis of the results of x-ray crystallographic analysis for zucchini ascorbate oxidase (40). The homologous region D (amino acids 546 -557 of DHGO) contains a cysteine, histidine, and methionine in positions homologous to the type-1 copper binding domain of small blue copper proteins, such as algal plastocyanin (41), azurin from Alcaligenes denitrificans (42), pseudoazurin from Alcaligenes faecalis (43), and plantacyanin from C. sativus (44).
Expression of Dihydrogeodin Oxidase Genomic DNA in P. frequentans-The cloned 5.5-kb SacI fragment, which contained whole DHGO genomic DNA, was first introduced into A. nidulans wild type strain FGSC 4. Although the integration of the introduced DNA fragment was confirmed by Southern blot analysis, no expression of DHGO polypeptide was detected by Western analysis (data not shown).
P. frequentans CMI 96659 possesses similar secondary metabolite biosynthetic pathway to that of A. terreus and produces asterric acid as an end product. The fungus has its own phenol oxidative coupling enzyme, sulochrin oxidase, 2 though whose nature such as molecular weight and immunoreactivity to anti-DHGO antibody is significantly different from DHGO. We introduced the cloned 5.5-kb SacI fragment (pBSDHGO) into P. frequentans by cotransformation with pDH25, hoping that the promoter of DHGO gene could function under similar secondary metabolism regulation in P. frequentans as that in A. terreus. All cotransformants obtained gave immunoreactive bands corresponding to the native DHGO protein by Western blot chain reaction amplification of the dihydrogeodin oxidase cDNA are overlined (P1, P2, P3, P4). N-terminal amino acid sequence obtained from the purified A. terreus dihydrogeodin oxidase is double-underlined in the amino acid sequence. Potential N-glycosylation sites are shown in boldface type. The polyadenylation site is indicated (*). analysis as shown in Fig. 6.
Expression of Dihydrogeodin Oxidase cDNA in A. nidulans-The cloned DHGO cDNA was ligated to fungal expression vector pTAex3. In this plasmid, expression of the cloned DHGO gene is controlled by the amyB promoter of A. oryzae (20). Expression of the gene under amyB promoter is strictly regulated by specific carbon source in culture medium, that is, induction by starch or maltose, and repression by glucose (20). A. nidulans was transformed with pTAexDHGO (cDNA in right direction, Fig. 7), pTAexDHGO-R (cDNA in opposite direction), or the vector plasmid pTAex3 as a control. Transformants were cultured first in glucose-containing medium for 3 days and then in starch-containing medium for a further 4 days to induce the expression. Proteins from the culture medium and mycelia were analyzed by Western blotting. Only in the mycelial extract of pTAexDHGO transformants was immunoreactive protein band with anti-DHGO antibody detected. No positive pro-teins were found in the culture medium or in mycelial extracts of control transformants with pTAex3 or pTAexDHGO-R (Fig.  8). The size of the recombinant DHGO expressed in A. nidulans is not distinguishable from that of A. terreus enzyme by Western blot analysis, suggesting that proper glycosylation occurred in A. nidulans.
The transcription level of the DHGO gene in representative transformants was examined by Northern blot analysis. The DHGO mRNA was detected when cultured under starch-induction conditions, although no detectable level of message was observed in non-induced cultures. Also, no message was detected in any of the controls (untransformant, transformants with pTAex3, or pTAexDHGO-R).
Product Identification-The product of the phenol oxidative coupling reaction mediated by the recombinant DHGO was identified by physico-chemical analysis. The HPLC retention time (Fig. 9), MS and NMR spectral data, and the specific optical rotation of the geodin formed by the recombinant DHGO was identical to that of authentic (ϩ)-geodin from A. terreus.

DISCUSSION
Dihydrogeodin oxidase catalyzes highly regio-and stereospecific phenol oxidative coupling of dihydrogeodin to form (ϩ)geodin. This is the first report of cloning of the cDNA and genomic DNA for the fungal multicopper blue protein catalyzing the specific phenol oxidative coupling reaction. The identity of the cloned gene for DHGO was confirmed by the following facts. 1) N-terminal amino acid sequence from the native DHGO was found in the deduced amino acid sequence of the cloned gene. 2) The expressed protein coded by the gene is immunoreactive with the specific polyclonal anti-DHGO IgG. 3) The expressed protein showed enzyme activity to form (ϩ)geodin from dihydrogeodin.
We have sequenced total 3041 bp of A. terreus genomic DNA, including 702 bp of the 5Ј-untranslated and 189 bp of the 3Ј-untranslated sequences. In the 702-bp region upstream from the translation initiation site, there are one TATA box-like sequence TATAAA and one CAAT box-like sequence CAAAT. This is consistent with the finding of similar putative eukaryotic regulatory sequences by Corden et al. (45). The cloned 5.5-kb genomic DNA of A. terreus possibly contained upstream regulatory sequence(s) for the expression of DHGO gene. Since DHGO expression was observed in P. frequentans transformants but not in A. nidulans transformants, P. frequentans might possess regulatory mechanism for the expression of secondary metabolism, asterric acid biosynthesis, analogous to that for (ϩ)-geodin biosynthesis in A. terreus, although A. nidulans might lack such a system.
In our previous study, DHGO was purified to be multicopper blue protein (9). In the deduced amino acid sequence of DHGO were found four regions homologous to regions strongly conserved among multicopper oxidases as shown in Fig. 5. The copper atoms bound in these domains are classified into three types corresponding to their distinct spectroscopic properties, type-1 (or blue), type-2 (or normal), and type-3 (or coupled binuclear) copper. Twelve potential ligands to these coppers were assigned on the basis of the results of x-ray crystallo-graphic analysis for zucchini ascorbate oxidase (40). Similarities between ascorbate oxidase and DHGO, the size (about 600 amino acids), solution structure (homodimer), and EPR spectra suggested that DHGO might contain four copper ions, one type-1, one type-2, and two type-3 coppers per subunit as is in ascorbate oxidase. Sequence alignment of these homologous regions of DHGO and other multicopper blue proteins indicated that the coordination sites for the three types of copper ions in DHGO are as follows. Type-1 copper ion coordinates at His-487, Cys-547, His-552, and Met-557. Type-2 copper ion coordinates at His-115 and His-490. Type-3 copper ion pair coordinates at His-117, His-162, His-164, His-494, His-546, and His-548 as shown in Fig. 5.
A catalytic mechanism for ascorbate oxidase has been proposed based on the available kinetic data, the three-dimensional structure, and the associated electron-transfer processes (46). Presence of the same type copper binding domains in DHGO suggests the involvement of a similar electron-transfer mechanism in the phenol oxidative coupling reaction catalyzed by DHGO. Type-1 copper is first reduced by one-electron transfer from the substrate dihydrogeodin, which is oxidized to a free radical. The electron is then transferred from type-1 copper to the type-3 copper pair. After reduction with four equivalents of reductant, the fully reduced enzyme is formed, which is able to bind dioxygen into the trinuclear copper center bridging the type-3 copper pair and the type-2 copper. This species must accept protons to release H 2 O, while intramolecular C-O coupling of dihydrogeodin diradicals gives (ϩ)-geodin under regioand stereospecific control of DHGO enzyme (Fig. 10). It is very interesting to make further study by site-directed mutagenesis of the DHGO gene and heterologous production of mutated DHGO proteins to conduct mechanistic investigation on electron transfer in multicopper blue protein and regio-and stereochemical control by the enzyme. The success of DHGO expression in A. nidulans makes this study possible.
DHGO catalyzes the final step of (ϩ)-geodin biosynthesis in A. terreus. Emodinanthrone, the initial cyclized biosynthetic precursor, is assumed to be elaborated by the specific polyketide synthase we call emodinanthrone synthase. However, no such anthrone-synthesizing enzyme activity has been detected ever in any of fungal cell-free systems. Recent studies suggested the cluster structure of genes for aflatoxin (47) and melanin (48) biosynthesis. By Northern blot analysis, two other message signals were detected by the cloned 5.5-kb SacI probe, indicating that two other enzymes are encoded along with DHGO in this 5.5-kb region. We are now analyzing whether these genes are involved in the biosynthesis of (ϩ)-geodin or not.