(Received for publication, May 15, 1995; and in revised form, July 6, 1995)
From the
Aspergillusterreus 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 Aspergillusnidulans 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 Penicilliumfrequentans 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 (+)-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
(+)-geodin in Aspergillus terreus (Fig. 1) at the
enzyme level (9, 10, 11, 12, 13) and
previously reported the purification of dihydrogeodin oxidase (DHGO) ()from A. terreus(9) , which catalyzes the
regio- and stereospecific formation of (+)-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, (
)although the clarified
properties were found to be quite different from those reported by
Nordlöv and Gatenbeck.
Figure 1: The biosynthesis of (+)-geodin in A. terreus.
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 peroxidase-like 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 multicopper blue enzymes in fungi.
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) . Aspergillusnidulans 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) .
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.
Genomic DNAs were subjected to restriction
enzyme digestion and transferred from agarose gel onto Nytran membrane
(Schleicher and Schuell) as described by Southern(26) . DNA
probes were labeled with digoxigenin-dUTP using DIG DNA labeling kit
(Boehringer Mannheim, Germany) according to the manufacturer's
instructions. Hybridization was carried out in a solution containing 5
SSC (1
SSC = 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 1% blocking reagent (Boehringer
Mannheim), 0.02% sodium N-lauroyl sarcosine at 60 °C
overnight. Filters were washed twice with 0.1
SSC, 0.1% SDS at
60 °C for 30 min. Enzyme-linked immunodetection was carried out
using DIG nucleic acid detection kit (Boehringer Mannheim) as
recommended by the manufacturer. Chemiluminescence detection with
3-(2`-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane,
disodium salt was also carried out.
The second cDNA library was constructed
using a TimeSaver cDNA Synthesis kit (Pharmacia) with
oligo(dT) primer. Packaging by Lambda Inn
packaging system (Nippon Gene, Japan) resulted in a library comprising
of 2
10
plaque-forming units, of which
approximately 98% were recombinants.
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 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
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).
Screening of the genomic DNA library (5
10
plaques) was carried out in the same way as that
of second cDNA library.
Figure 3: Nucleotide and deduced amino acid sequences of A. terreus dihydrogeodin oxidase. Putative TATA and CAAT boxes are underlined in the 5`-flanking region. Copper binding sites are boxed. Introns are underlined. Sequences of oligonucleotides used in the polymerase 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 boldfacetype. The polyadenylation site is indicated (*).
A rapid amplification of cDNA ends
method (28) was used to extend the 5`-end of DHGO cDNA. One
microgram of poly(A) 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
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.
Figure 2: Restriction enzyme map of the cloned genomic DNA and cDNA encoding dihydrogeodin oxidase of A. terreus. A, genomic DNA; B, cDNA. cDNAs 1-4 were obtained by screening the second cDNA library; cDNA 5 was cloned by the rapid amplification of cDNA ends method. Blackboxes represent the coding regions for dihydrogeodin oxidase, and whiteboxes indicate introns. Only major restriction enzyme sites are shown.
A partial genomic DNA library of
size-fractionated SacI-digested 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.
Sequence analysis of the 5`-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 glycosylation 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 N-glycosylation in the mature DHGO protein.
Figure 4: SDS-PAGE analysis of the purified dihydrogeodin oxidase from A. terreus. Lane1, dihydrogeodin oxidase digested with N-glycanase; lane2, untreated dihydrogeodin oxidase; M, molecular weight marker.
Significant homology was observed with multicopper oxidases, including fungal laccase genes from Neurospora crassa(35) , Coriolus hirsutus (36), Cryphonectria parasitica(37) , cucumber (Cucumis sativus)ascorbate oxidase(38) , and human ceruloplasmin(39) . The degrees of amino acid sequence identity between A. terreus DHGO and laccase of N. crassa(35) and ascorbate oxidase of C. sativus(38) are 36 and 29%, respectively.
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) .
Figure 5: Amino acid sequence comparison of copper binding motifs of the multicopper blue proteins. The boxedregionsA, B, C, and D, aligned according to the study by Ohkawa et al.(38) , contain 12 potential ligands of type-1, type-2, and type-3 copper on the basis of the results of x-ray crystallographic analysis for zucchini ascorbate oxidase(40) . Sequences of dihydrogeodin oxidase (DHGO) from A. terreus, laccase (LAC) from N. crassa, ascorbate oxidase (ASO) from cucumber updated using the Brookhaven protein data base, and human ceruloplasmin (CP) are shown. The number on the left of each sequence represents the position of the amino acid residues of the protein. Identical amino acids are boxed. The numbers1, 2, and 3 indicate potential coordination sites for three different types of copper ions.
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, 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 analysis as
shown in Fig. 6.
Figure 6: Immunological detection of dihydrogeodin oxidase expressed in P. frequentans by immunoblotting. The mycelial protein fractions were separated by SDS-polyacrylamide gel electrophoresis. After blotting to the nitrocellulose filter, immunological detection with the polyclonal antibodies against dihydrogeodin oxidase was carried out. The samples are as follows: laneA, crude proteins from P. frequentans wild type; lanesT1-T5, crude proteins from P. frequentans transformants; laneB, 0.1 µg of purified dihydrogeodin oxidase.
Figure 7: Expression plasmid pTAexDHGO. The dihydrogeodin oxidase cDNA is shown as an openbox. The EcoRI fragment of dihydrogeodin oxidase cDNA cloned in pT7 Blue vector was ligated into pTAex3 at the EcoRI site to construct pTAexDHGO. The promoter of A. oryzae Taka-amylase A (amyB) directs the starch or maltose-inducible expression of dihydrogeodin oxidase(20) .
Figure 8: Analysis of dihydrogeodin oxidase derived from recombinant A. nidulans by immunoblotting. The concentrated supernatant and the mycelial protein fractions of A. nidulans transformants were separated by SDS-polyacrylamide gel electrophoresis. After blotting to the nitrocellulose filter, immunological detection with the polyclonal antibodies against dihydrogeodin oxidase was carried out. The samples are as follows: laneB, 0.05 µg of dihydrogeodin oxidase; lanes1-3, 20 µl of the mycelial fractions from the pTAexDHGO transformants 1-3, respectively; lane4, 20 µl of mycelial fractions derived from the pTAexDHGO-R transformants; lane5, 20 µl of the concentrated culture liquid from pTAexDHGO transformant 1; laneA, 0.1 µg of dihydrogeodin oxidase.
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).
Figure 9: HPLC analysis of the recombinant DHGO reaction product. A, reaction product with the recombinant enzyme from pTAexDHGO transformant; B, reaction product with the recombinant enzyme from pTAexDHGO-R transformant.
Dihydrogeodin oxidase catalyzes highly regio- and stereospecific phenol oxidative coupling of dihydrogeodin to form (+)-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 (+)-geodin from dihydrogeodin.
We have sequenced total 3041 bp of A. terreus genomic DNA, including 702 bp of the 5`-untranslated and 189 bp of the 3`-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 (+)-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 crystallographic 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 HO, while intramolecular C-O
coupling of dihydrogeodin diradicals gives (+)-geodin under regio-
and 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.
Figure 10: Postulated catalytic mechanism for formation of (+)-geodin from dihydrogeodin by phenol oxidative coupling involving two one-electron oxidation steps.
DHGO catalyzes the final step of (+)-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 (+)-geodin or not.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49538[GenBank].