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Originally published In Press as doi:10.1074/jbc.M701239200 on March 20, 2007

J. Biol. Chem., Vol. 282, Issue 19, 14476-14481, May 11, 2007
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Pentaketide Resorcylic Acid Synthesis by Type III Polyketide Synthase from Neurospora crassa*Formula

Nobutaka Funa, Takayoshi Awakawa, and Sueharu Horinouchi1

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

Received for publication, February 9, 2007 , and in revised form, March 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Type III polyketide synthases (PKSs) are responsible for aromatic polyketide synthesis in plants and bacteria. Genome analysis of filamentous fungi has predicted the presence of fungal type III PKSs, although none have thus far been functionally characterized. In the genome of Neurospora crassa, a single open reading frame, NCU04801.1, annotated as a type III PKS was found. In this report, we demonstrate that NCU04801.1 is a novel type III PKS catalyzing the synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named 2'-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a starter substrate and condensed four molecules of malonyl-CoA to give a pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic acids, aldol condensation and aromatization of the intermediate, which is still attached to the enzyme, are presumably followed by hydrolysis for release of the product as a resorcylic acid. ORAS is the first type III PKS that synthesizes pentaketide resorcylic acids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyketides are synthesized by so-called polyketide synthases (PKSs)2 that fall into three groups, types I to III (1). Ketosynthase, which is a pivotal domain for polyketide synthesis, catalyzes the sequential decarboxylative condensation of extender substrates by a process closely similar to fatty acid biosynthesis. In contrast to type I and II megasynthases composed of ketosynthases and accessory enzymes, type III PKSs are a dimer of ketosynthase that accomplishes a complex set of reactions, such as priming of a starter substrate, decarboxylative condensation of extender substrates, ring closure, and aromatization of the polyketide chain, in a multifunctional active site pocket (2). A growing number of type III PKSs catalyzing the synthesis of aromatic polyketides have been found from plants and bacteria, and their catalytic properties have been characterized. Chalcone synthases, the representative of type III PKSs in plants, catalyze the synthesis of naringenin chalcone, which is a common precursor of all flavonoids produced by plants (2). RppA, the first type III PKS found in bacteria, catalyzes the synthesis of 1,3,6,8-tetrahydroxynaphthalene, which is a precursor of hexahydroxyperylenequinone melanin in a bacterium, Streptomyces griseus (3).

Filamentous fungi produce a vast array of polyketides such as melanins, antibiotics, and mycotoxins (4). The pathways and regulation of biosynthesis of the aromatic polyketides in filamentous fungi have been studied extensively for mycotoxins, including aflatoxin and fumonisin, produced by food spoilage fungi (5) and for melanogenesis associated with infection of plant-pathogenic fungi (6). In addition, the clinical benefit of lovastatin, a cholesterol-lowering agent, produced by Aspergillus terreus (7), promotes isolation of the biosynthetic genes for polyketides in fungi. Nevertheless, the enzyme systems for the synthesis of fungal polyketides have been confined to iterative type I PKSs, which are composed of a large multifunctional polypeptide containing discrete functional domains (1). This makes sense from an evolutionary point of view, because the iterative type I PKS system is structurally and mechanistically similar to the enzyme system of fatty acids synthesis in fungi and yeast (8).

On the other hand, recent genome projects for Neurospora crassa (9) and Aspergillus oryzae (10) predict the presence of type III PKSs in these filamentous fungi (11). Type III PKSs share an evolutionary origin with the type II fatty acid synthases in plants and bacteria. N. crassa contains a single gene, NCU04801.1, which is thought to encode a type III PKS. In the present study, we characterized the gene product of NCU04801.1, which was found to actually encode a type III PKS. NCU04801.1 turned out to be a novel type III PKS that catalyzes 2'-oxoalkylresorcylic acid synthesis from a long-chain fatty acid CoA ester and four molecules of malonyl-CoA (Fig. 1). We hence named NCU04801.1 ORAS (2'-oxoalkylresorcylic acid synthase). This is the first report that describes the catalytic property of a fungal type III PKS and the synthesis of a pentaketide alkylresorcylic acid by type III PKSs in nature.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Escherichia coli strains JM109 and BL21(DE3), plasmid pUC19, restriction enzymes, T4 DNA ligase, and TaqDNA polymerase were purchased from Takara Biochemicals (Shiga, Japan). pET26b was purchased from Novagen (San Diego, CA). An N. crassa conidial cDNA library and N. crassa strains FGSC 9717, 9718, 9719, and 9720 were purchased from the Fungal Genetic Stock Center, University of Kansas Medical Center, Kansas City. [2-14C]Malonyl-CoA was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Nonlabeled CoA esters and 2-carboxymethyl-4,6-dimethoxy-benzoic acid were purchased from Sigma. Stearoyl chloride was purchased from Aldrich.


Figure 1
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  		FIGURE 1.
Summary of reactions catalyzed by ORAS as a 2'-oxoalkylresorcylic acid synthase. Decarboxylation of unstable resorcylic acids, resulting in formation of the corresponding resorcinols, occurs non-enzymatically.

 
Construction of pET26b-ORAS—Using the N. crassa conidial cDNA library prepared from the conidium stage as a template, a 1.3-kb DNA fragment containing the NCU04801.1-coding region was amplified by PCR with primer I (5'-GCGGAATTC-CATATGGCTGCTTCAACATTGTCCAA-3', with an EcoRI site shown by underlining, an NdeI site shown by the italic letters, and the start codon of NCU04801.1 shown by the boldface letters) and primer II (5'-GCGAAGCTTATCCAGTTCAACATTGTCCAACG-3', with a HindIII site shown by underlining). The amplified fragment was cloned between the EcoRI and HindIII sites of pUC19, resulting in pUC19-ORAS. The NdeI-HindIII fragment containing the NCU04801.1 sequence was excised from pUC19-ORAS and cloned between the NdeI and HindIII sites of pET26b, resulting in pET26b-ORAS.

Production and Purification of Histidine-tagged ORAS—For production of histidine-tagged ORAS, E. coli BL21(DE3) harboring pET26b-ORAS was grown overnight in Luria broth containing 100 µg/ml kanamycin. Cells were harvested by centrifugation, resuspended in buffer containing 10 mM Tris-HCl (pH 8.0) and 10% glycerol, and disrupted by sonication. A crude cell-lysate was prepared by removal of cell debris by centrifugation at 10,000 x g for 20 min. ORAS was purified by using a nickel-nitrilotriacetic acid spin column (Qiagen) according to the manual from the manufacturer. The purified histidine-tagged protein was dialyzed against 10 mM Tris-HCl (pH 8.0) and 10% glycerol.

PKS Assay—The standard reaction mixture contained 100 µM [2-14C]malonyl-CoA, 100 µM starter-CoA, 100 mM Tris-HCl (pH 7.0), and 3.9 µg of ORAS in a total volume of 100 µl. Reaction was incubated at 30 °C for 30 min before being quenched with 20 µl of 6 M HCl. The products were extracted with ethyl acetate, and the organic layer was evaporated to dryness. The residual material was dissolved in 20 µl of methanol for thin-layer chromatography (TLC) and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) analysis. Silica gel 60 WF254 TLC plates (Merck) were developed in benzene/acetone/acetic acid (85: 15:1, v/v/v) and the 14C-labeled compounds were detected by using a BAS-MS imaging plate (Fuji Film).

LC-APCIMS analysis was carried out by using the esquire HCT system (Bruker Daltonics) equipped with a Pegasil-B C4 reversed-phase HPLC column (4.6 x 250 mm; Senshu Scientific, Tokyo). The compounds were eluted with a linear gradient of 70 to 100% CH3CN in water (each containing 0.1% acetic acid) at a flow rate of 1 ml/min. Triketide pyrones [2b]to[2i], tetraketide pyrones [3c]to[3f], and tetraketide resorcinols [5a] to [5f] were identified by comparing their LC-MS and MS/MS spectra with those obtained from the authentic standards that had been prepared by ArsB and ArsC, type III PKSs from Azo-tobacter vinelandii (12).


Figure 2
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  		FIGURE 2.
Product identification by LC-MS/MS analysis. A, structure of 2,4-dimethoxy-6-(2'-oxononadecyl)-benzoic acid, a methyl ether of 6b. A lactol tautomeric form is also shown. B, LC-MS/MS spectrum of the methylated compound derived from 2,4-dihydroxy-6-(2'-oxononadecyl)-benzoic acid [6b]. A rhomboid indicates a parent ion of MS/MS analysis. Inset is an extracted ion chromatogram of negative LC-APCIMS at m/z = 461. C, LC-MS/MS spectrum of chemically synthesized 2,4-dimethoxy-6-(2'-oxononadecyl)-benzoic acid. Insets in B and C are extract ion chromtograms of negative LC-APCIMS at m/z = 461.

 
For identification of a pentaketide resorcylic acid, 2,4-dihydroxy-6-(2'-oxononadecyl)-benzoic acid [6b] was converted to a stable methyl ether, 2,4-dimethoxy-6-(2'-oxononadecyl)-benzoic acid. The spectrometric data of the methyl ether was compared with those of the synthetic standard. 6b was methylated as follows. To a stirring acetone solution of 6b, which was prepared by ORAS in a 50-ml scale reaction, were added 100 mg of anhydrous K2CO3 and an excess amount of CH3I, and then the mixture was refluxed for 4 h. After K2CO3 had been filtered off, the mixture was evaporated, dissolved in ethyl acetate, and washed with 1 M HCl and brine. The organic phase was concentrated under vacuum to give a sample for LC-MS analysis. Extracted ion chromatograms and LC-MS/MS spectra are shown in Fig. 2. Spectral data are summarized in supplemental Table 1.

Synthesis of 2,4-Dimethoxy-6-(2'-oxononadecyl)-benzoic Acid—2,4-Dimethoxy-6-(2'-oxononadecyl)-benzoic acid was synthesized as described by Saeed (13). A stirring mixture of 30 mg (0.125 mmol) of 2-carboxymethyl-4,6-dimethoxy-benzoic acid and 152 mg (0.501 mmol) of stearoyl chloride was heated in an oil bath at 200 °C for 4 h. Preparative TLC developed in hexane/ethyl acetate (5:1, v/v) gave 3-heptadecyl-6,8-dimethoxy-isochromen-1-one (6 mg, 11% yield) as a white solid: 1H NMR (500 MHz, CDCl3) {delta} 0.86 (t, J = 7.0 Hz, 3H, C17'H), 1.25 (m, 28H, methylene of C3' to C16'), 1.65 (m, 2H, C2'H), 2.43 (t, J = 7.0 Hz, 2H, C1'H), 3.88 (s, 3H, CH3O-C6), 3.94 (s, 3H, CH3O-C8), 6.06 (s, 1H, C4H), 6.30 (d, J = 2.0 Hz, 1H, C5H), 6.40 (d, J = 2.0 Hz, 1H, C7H). To a solution of 4 mg (0.009 mmol) of 3-heptadecyl-6,8-dimethoxy-isochromen-1-one in 3 ml of ethanol was added 6 ml of 5% KOH solution, and the mixture was refluxed for 4 h. After the mixture had been cooled at ambient temperature, the ethanol was removed by evaporation. A small amount of water was added, and the mixture was acidified with diluted HCl and extracted with ethyl acetate. The ethyl acetate phase was washed with brine and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the organic layer was evaporated under reduced pressure. Preparative TLC developed in hexane/ethyl acetate/acetic acid (50: 60:1, v/v/v) gave 2,4-dimethoxy-6-(2'-oxononadecyl)-benzoic acid (4 mg, 96% yield) as a white solid: 1H NMR (500 MHz, CDCl3) {delta} 0.86 (t, J = 7.0 Hz, 3H, C19'H), 1.26 (m, 30H, methylene of C4' to C18'), 1.59 (m, 2H, C3'H), 2.32 (t, J = 7.5 Hz, 1H, C4H of tautomeric lactol form; see Fig. 2A), 2.59 (t, J = 7.5 Hz, 1H, C4H of lactol tautomeric form), 3.84 (s, 3H, CH3O-C2), 3.99 (s, 3H, CH3O-C4), 4.04 (s, 2H, C1'H), 6.39 (d, J = 2.0 Hz, 1H, C3H), 6.47 (d, J = 2.0 Hz, 1H, C5H).

Determination of Kinetic Parameters of ORAS—The 100-µl scale reactions contained 100 mM Tris-HCl (pH 7.0), 100 µM [2-14C]malonyl-CoA (19,800 dpm), and 0.99, 0.51, or 0.25 µM ORAS for the hexanoyl-CoA-, lauroyl-CoA-, or stearoyl-CoA-primed reaction, respectively. The concentrations of starter-CoAs were varied between 3.0 to 15, 0.5 to 1.75, and 0.5 to 1.75 µM for hexanoyl-CoA, lauroyl-CoA, and stearoyl-CoA, respectively. After the reaction mixture had been preincubated at 30 °C for 4 min, the reactions were initiated by adding the substrates. The reactions were continued for 300, 60, or 45 s for hexanoyl-CoA, lauroyl-CoA, or stearoyl-CoA, respectively. The reactions were stopped with 20 µl of 6 M HCl, and the material in the mixture was extracted with ethyl acetate. The organic layer was evaporated and combined with nonlabeled carrier products for HPLC analysis. After separation by HPLC, the amounts of polyketides were quantified by means of [2-14C]malonyl-CoA incorporation. Steady-state parameters were determined by Lineweaver-Burk plots.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transcription of the ORAS Gene in N. crassa—In this study, we used the cDNA library prepared from the conidium stage of N. crassa for cloning the ORAS gene. We determined whether the ORAS gene was also transcribed during the mycelium and perithecium stages. Reverse transcription-PCR experiments, using the commercially available cDNA libraries prepared for the mycelium, conidium, and perithecium stages, all gave a distinct PCR product of the same length in almost the same amount (data not shown), indicating that the ORAS gene is expressed throughout growth. We also disrupted the ORAS gene on the chromosome of strains FGSC 9717, 9718, 9719, 9720, having a mutation in the KU genes (14), by inserting a hygromycin B resistance gene into the ORAS-coding region. KU was originally identified as an autoantigen recognized by sera and later was found to be involved in nonhomologous recombination in eukaryotes (14). True disruptants, which were checked by Southern hybridization, were obtained after repeated growth of hygromycin-resistant colonies on agar medium containing 500 µg/ml hygromycin B. These disruptants grew normally and differentiated to form conidia on several agar media (data not shown), which showed that ORAS mutations gave no apparent phenotypic changes. We could not determine the loss of the ability of the disruptants to produce alkylresorcylic acids and alkylresorcinols (the products of ORAS; see below), because the parental strains produced these polyketides in such a small amount that we failed to detect production by HPLC.


Figure 3
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  		FIGURE 3.
Radio-TLC analysis of products synthesized by ORAS from various acyl-CoA starter substrates and [2-14C]malonyl-CoA. The starter substrates used were acetyl-CoA [1j](lane 2), butyryl-CoA [1i](lane 4), hexanoyl-CoA [1h](lane 6), octanoyl-CoA [1g](lane 8), decanoyl-CoA [1f](lane 10), lauroyl-CoA [1e](lane 12), myristoyl-CoA [1d](lane 14), palmitoyl-CoA [1c] (lane 16), stearoyl-CoA [1b](lane 18), arachidoyl-CoA [1a](lane 20), and p-coumaroyl-CoA (lane cou). Lane m is a control incubation without a starter substrate.

 


Figure 4
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  		FIGURE 4.
Time course of 2,4-hydroxy-6-(2'-oxononadecyl)-benzoic acid [6b] formation by ORAS. The molecular weights of resorcinol 7b and 6b are 390 and 434, respectively. The solid line, representing 7b, and the dashed line, representing 6b, indicate extracted ion chromatograms of negative LC-APCIMS at m/z = 389 and 433, respectively. Because of the instability of 6b, decarboxylation of 6b to give 7b occurred at the ionization step of mass spectrometry after they had been separated by HPLC. This is why the ion peak representing 7b (m/z = 389) was also detected at the retention time corresponding to 6b (m/z = 433).

 
Production of His-tagged ORAS in E. coli—The amino acid alignment of ORAS with plant and bacterial type III PKSs (supplemental Fig. 1) showed that the Cys-His-Asn catalytic triad, which is required for priming and extension of polyketides in type III PKSs (2), was conserved in ORAS. We constructed an E. coli plasmid to prepare ORAS with a His tag at its C terminus and to determine its in vitro catalytic properties. The plasmid we constructed was pET26b-ORAS, which contained the ORAS gene under the control of the T7 promoter. This plasmid would direct the synthesis of Met-ORAS (Ala2-Asp445)-Lys-Leu-Ala3-Leu-Glu-His6. The histidine-tagged ORAS protein was purified by using a nickel-nitrilotriacetic acid column. The purified ORAS protein gave a single protein band at a position of ~48 kDa on SDS-PAGE (data not shown).

In Vitro Analysis of ORAS Reactions—We first checked whether ORAS accepts p-coumaroyl-CoA, a substrate of chalcone synthase, as a starter substrate by incubating ORAS in the presence of p-coumaroyl-CoA and malonyl-CoA as an extender substrate. No products were observed (Fig. 3), in agreement with the idea that filamentous fungi produce no flavonoid-related compounds. We then tested whether ORAS could accept CoA esters of C2 to C20 straight-chain fatty acids as a starter substrate, because the fatty acids and their intermediates are abundant primary metabolites in N. crassa (15) and because ArsB, a type III PKS in A. vinelandii, produces alkylresorcinols by using long-chain fatty acid CoA esters as starters and malonyl-CoA as extenders (12). [2-14C]Malonyl-CoA was used as an extender substrate to follow reaction products by TLC. As we expected, ORAS yielded 14C-labeled products for a broad range of CoA esters of C4-C20 fatty acids (Fig. 3).


Figure 5
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  		FIGURE 5.
A proposed mechanism of ORAS reaction to produce alkylresorcylic acids and alkylresorcinols. A, alkylresorcinol synthesis by ArsB (12). B, alkylresorcylic acid synthesis by ORAS. Hydrolysis of the thioester takes place after aldol condensation and aromatization, and therefore the carboxyl group remains to the product. C, alkylresorcinol synthesis by hydrolysis followed by decarboxylative aldol condensation and aromatization. D, synchronous synthesis of alkylresorcylic acid and alkylresorcinol by hydrolysis followed by aldol condensation and aromatization. The dashed line denotes an unlikely route in which aldol condensation and hydrolysis occur before aromatization.

 
The products were identified as described under "Experimental Procedures." Their chemical structures, together with their biosynthesis routes, are illustrated in Fig. 1. ORAS gave triketide pyrones [2b]to[2i] from the C4 to C18 substrates, as revealed by radio-TLC (Fig. 3) and LC-APCIMS analysis (Supplementary Table 1). In addition, tetraketide resorcinols [5a]to [5f] were produced from the C10-C20 substrates and tetraketide pyrones [3c]to[3f] were also produced from the C10 to C16 substrates (Fig. 3). Interestingly, ORAS produced pentaketide resorcinols [7a]to[7c] from the C16-C20 substrates, pentaketide resorcylic acids [6a] and [6b] from the C18-C20 substrates, and tetraketide resorcylic acid [4a] from the C20 substrate [1a]. The structure of tetraketide resorcylic acid [4a] was deduced from the observed [M - H]- ion (supplemental Table 1). The structure deduction was supported by the observation that 4a was non-enzymatically converted to a tetraketide resorcinol [5a]. The chemical structures of pentaketide resorcylic acids were elucidated by a comparison of LC-MS/MS spectra between the methyl ether of 6b with the chemically synthesized authentic sample (Fig. 2) as described under "Experimental Procedures."

We observed that ORAS produced the pentaketide resorcylic acid [6b] exclusively when the reaction period was shortened. The prolonged reaction resulted in a decrease of the pentaketide resorcylic acid [6b] with a concomitant increase of the pentaketide resorcinol [7b] (Fig. 4). Therefore we concluded that the resorcinols produced by the action of ORAS resulted from non-enzymatic decarboxylation of the corresponding resorcylic acids (see Discussion). Hence, ORAS is the first type III PKS that catalyzes the synthesis of a pentaketide resorcylic acid, 2'-oxoalkylresorcylic acid.

Kinetic Analysis of ORAS—The product profile shown in Fig. 3 was not affected by pH change from 6 to 8. The pH optimum, as determined by measuring consumption of hexanoyl-CoA [1h], lauroyl-CoA [1e], and stearoyl-CoA [1b], was around pH 7. ORAS gave no products when the pH was lower than 5. We chose 1h, 1e, and 1b as representatives for measuring the steady state kinetic parameters of ORAS. The kcat/Km value for stearoyl-CoA [1b] consumption was the largest among these substrates (Table 1), indicating that ORAS prefers the CoA esters derived from long-chain fatty acids. In addition, the largest kcat/Km value in the polyketide formation was observed for the pentaketide resorcylic acid [6b] synthesis (Table 2). Considering the observation that the primary products of ORAS were resorcylic acids, we conclude that ORAS is a novel type III PKS that is responsible for the synthesis of 2'-oxoalkylresorcylic acid from long-chain starter substrates.


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  		TABLE 1
Steady-state kinetic parameters of the starter-CoA consumption by ORAS

Results are mean ± S.E. (n = 3).

 


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  		TABLE 2
Steady-state kinetic parameters of the ORAS reactions

Results are mean ± S.E. (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The product profile of ORAS can be categorized into three groups: (i) triketide pyrone synthesis from C4,C6, and C8 starter units; (ii) triketide pyrone synthesis, accompanied by tetraketide resorcinol and tetraketide pyrone synthesis in small amounts, from C10,C12, and C14 starter units; and (iii) tetraketide and pentaketide resorcylic acid synthesis from C16,C18, and C20 starter units. It is evident that the incorporation of longer starter substrates leads to an increased extension time of the extender substrate. We assume that the increased extension time results from stable retention of the hydrophobic substrates in the active site of ORAS. Recently, a hydrophobic tunnel, which accounts for binding of long-chain aliphatic substrates, has been found in the structure of PKS18, a type III PKS responsible for pyrone synthesis from C12 to C20 starter units (16). A similar tunnel may also exist in ORAS, although ORAS differs from PKS18 in that it prefers stearoyl-CoA among hexanoyl-CoA, lauroyl-CoA, and stearoyl-CoA (Table 1), whereas PKS18 prefers lauroyl-CoA (17).

ORAS is the first type III PKS that catalyzes the formation of pentaketide resorcylic acids, although there are many type III PKSs that produce tetraketide resorcinols, such as stilbene (18), stilbene carboxylic acid (19), and alkylresorcinol (12). Another unique feature of ORAS is the ability to synthesize resorcylic acids. This finding suggests a mechanistic difference between ORAS and ArsB, which shares 24% amino acid similarity with ORAS. Considering that ArsB produces alkylresorcinols exclusively (12), we assume that ArsB synthesizes a resorcinol scaffold by decarboxylative aldol condensation (Fig. 5A). In contrast, in the ORAS reaction, aldol condensation and aromatization presumably take place before hydrolysis, resulting in producing the resorcylic acid scaffold (Fig. 5B). If the hydrolysis precedes aldol condensation, two cases for synthesis of the resorcinol scaffold are possible: one, decarboxylative aldol condensation (Fig. 5C), and the other, aldol condensation, which does not accompany decarboxylation (Fig. 5D). In the latter case, retention or detachment of the carboxylic acid depends on whether or not decarboxylation is concerted with aromatization (Fig. 5D). However, these are unlikely possibilities for the ORAS reaction, because these reactions would lead to the production of alkylresorcinol, which is not a direct product of ORAS.

Although Neurospora is a model organism for studying the genetics, biochemistry, and molecular biology of filamentous fungi, no reports describing secondary metabolism in this genus have been published. It is not clear whether alkylresorcylic acids and alkylresorcinols in fungi play a role as membrane components as found for the cysts of A. vinelandii (12) or are just secondary metabolites. 2'-Oxoalkylresorcinol is known to be a metabolite of a basidiomycete (20), rice (21) and wheat and rye (22), suggesting the occurrence of type III PKSs with ORAS activity in these organisms. It is also possible that ORAS-like enzymes are involved in the synthesis of some tetraketide alkylresorcinols in other fungi, such as Aspergillus (23) and Fusarium (24).


   FOOTNOTES
 
* This work was supported by Research Grant Program 2005 of the New Energy and Industrial Technology Development Organization of Japan (05A07510d) and a grant-in-aid for scientific research on priority areas "Applied Genomics" from Monkasho. 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Table 1. Back

1 To whom correspondence should be addressed. Tel.: 81-3-5841-5123; Fax: 81-3-5841-8021; E-mail: asuhori{at}mail.ecc.u-tokyo.ac.jp.

2 The abbreviations used are: PKS, polyketide synthase; HPLC, high-performance liquid chromatography; LC, liquid chromatography; APCIMS, atmospheric pressure chemical ionization mass spectrometry; ORAS, 2'-oxoalkylresorcylic acid synthase; MS/MS, tandem mass spectrometry. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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