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Eukaryotic Cell, March 2007, p. 514-520, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00226-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities{triangledown}

Francisco Ferrer-Sevillano and José M. Fernández-Cañón*

Instituto de Biología Molecular, Genómica y Proteómica, Universidad de León, Campus de Vegazana, s/n 24071 León, Spain

Received 13 July 2006/ Accepted 27 November 2006


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ABSTRACT
 
Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain ({Delta}phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).


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INTRODUCTION
 
Most living organisms use a small number of catabolic pathways to obtain energy and structural components from organic materials. However, some microorganisms have been able to organize several catabolic pathways in order to grow by using uncommon compounds (of natural or artificial origin) as a carbon source, thereby contributing to the recycling of many of these compounds.

Phenylacetic acid (PhAc) and its hydroxyderivatives (OH-PhAc) are aromatic compounds catabolized by particular organisms, mostly bacteria and fungi, by the following three different pathways (1, 6, 13, 15): (i) the 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) or homogentisic acid pathway, (ii) the 3,4-dihydroxyphenylacetate (3,4-diOH-PhAc) or homoprotocatechuate pathway, and (iii) the phenylacetyl-coenzyme A (PhAc-CoA) pathway.

Usually, the same microorganism catabolizes PhAc and its hydroxyderivatives by using several pathways. For example, Pseudomonas putida catabolizes PhAc via a phenylacetyl-CoA pathway, 3-hydroxyphenylacetate via a 2,5-diOH-PhAc pathway, and 4-dihydroxyphenylacetate via a 3,4-dihydroxyphenylacetic acid pathway (2). This implies the production of enzymes for three full pathways to catabolize just three compounds and does not appear to be a very efficient way to use the cellular machinery.

Aspergillus nidulans is a filamentous fungus which is able to grow in PhAc and PhAc-hydroxyderivatives as the only carbon source by using the 2,5-diOH-PhAc pathway. There are some microorganisms which catabolize PhAc through the 2,5-diOH-PhAc pathway (mainly via 3-hydroxyphenylacetate) (1, 18, 19), but in A. nidulans, this catabolic pathway occurs via 2-hydroxyphenylacetate (6, 7, 10). PhAc is converted to 2,5-diOH-PhAc through two sequential hydroxylations in the aromatic ring, at positions 2 and 5 (Fig. 1). In addition, this fungus is a eukaryotic organism which, unlike bacteria, uses cytochromes P450 to hydroxylate the aromatic rings of these compounds.


Figure 1
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  		FIG. 1. Catabolic pathways of phenylalanine, tyrosine, phenylacetate, and mono-, di-, and trihydroxyphenylacetate derivatives in A. nidulans. Enzymes involved in the degradation of phenylalanine to fumarate and acetoacetate are present in A. nidulans and humans. Enzymes required to catabolize phenylacetate and hydroxyderivatives to homogentisate are present in A. nidulans (and some microorganisms). Enzymes: 1, phacA-encoded phenylacetate 2-hydroxylase (also catalyzes, to a lesser extent, 3-hydroxyphenylacetate 6-hydroxylation); 2, 2-hydroxyphenylacetate 5-hydroxylase; 3, phenylalanine hydroxylase; 4, tyrosine aminotransferase; 5, 4-hydroxyphenylpyruvate dioxygenase; 6, homogentisate dioxygenase; 7, maleylacetoacetate isomerase; 8, fumarylacetoacetate hydrolase; 9, phacB-encoded 3-hydroxyphenylacetate 6-hydroxylase (also catalyzes, to a lesser extent, phenylacetate 2-hydroxylation); 10, 4-hydroxyphenylacetate 3-hydroxylase. Note the change in carbon numbers after hydroxylation.

The homogentisate pathway is also used to catabolize phenylalanine and tyrosine. Upper pathways are specific for PhAc (and hydroxyderivatives) and phenylalanine/tyrosine, and the lower pathway (from 2,5-diOH-PhAc to fumarate and acetoacetate) is common to PhAc and phenylalanine/tyrosine catabolism (6, 10) (Fig. 1).

Previously, by using cDNA subtraction, we were able to clone several genes induced by PhAc from A. nidulans and use them to clone the corresponding human and mouse genes (4-8, 10). One of them, phacA, was the first member of a new family of cytochromes P450 (CYP504A1) and catalyzes the 2-hydroxylation of PhAc. Loss-of-function mutation of phacA results in residual growth on PhAc as the only carbon source, but the fungus retains some capacity to produce 2-hydroxyphenylacetic acid, which indicates that more than one gene is responsible for 2-hydroxylation of PhAc. We found a second gene involved in 2-hydroxylation of PhAc (10). This second gene, previously denoted pshA and now called phacB, also encodes a new cytochrome P450, which defines a new subfamily (CYP504B). In this work, we show that phacB codifies a 3-hydroxy-PhAc and 3,4-dihydroxy-PhAc 6-hydroxylase and catalyzes the 6-hydroxylation of 3-hydroxyphenylacetate to produce 2,5-dihydroxy-PhAc (homogentisic acid) and of 3,4-dihydroxy-PhAc to produce 2,4,5-trihydroxy-PhAc. This new cytochrome P450 can also catalyze, to a lesser extent, the 2-hydroxylation of PhAc to produce 2-hydroxy-PhAc. Here we also report gas chromatography-mass spectrometry (GC-MS) characterization of the 2,4,5-trihydroxyphenylacetate synthesized in vitro by the cytochrome P450 product of the phacB gene.

Functional characterization of the phacB gene is not only important for the catabolism of phenylacetic acid but also for penicillin biosynthesis, because PhAc is a moiety of the penicillin G molecule. Penicillium chrysogenum, the industrial producer of penicillin, transforms PhAc into 2-hydroxyphenylacetate, which is not suitable for penicillin production in the fermentation broth, decreasing the available PhAc for penicillin biosynthesis. For this reason, a PhAc hydroxylation mutant of P. chrysogenum was long sought but never found. After the cloning of phacA, we showed that by decreasing the PhAc hydroxylating capacity, we could increase penicillin production in A. nidulans (10). Recently, it was shown that P. chrysogenum, after having lost its PhAc hydroxylating capacity, also increases penicillin production fivefold (14).

For these reasons, the characterization of phacB has academic, industrial, and ecological interest.


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MATERIALS AND METHODS
 
Strains, media, and growth conditions. The Aspergillus nidulans strains used in this work are as follows. The biA1 strain, a biotin auxotroph, was used as a wild type (6, 7). The {Delta}hmgA (argB+::hmgA) biA1 methG1 mutant has a disrupted hmgA gene, is homogentisate dioxygenase deficient, and is a biotin and methionine auxotroph (7). The {Delta}phacA (argB+::phacA) biA1 methG1 mutant has a disrupted phacA gene, is PhAc 2-hydroxylase deficient, and is a biotin and methionine auxotroph (10). The {Delta}phacB (argB+::phacB) biA1 methG1 mutant has a disrupted phacB gene and is a biotin and methionine auxotroph created for this study. The {Delta}phacA {Delta}phacB biA1 double mutant has disrupted phacA and phacB genes and is a biotin auxotroph created for this study. The biA1 methG1 argB2 strain is a biotin, methionine, and arginine auxotroph and was used as a recipient for phacB gene disruption (6, 7, 10). The biA1 methG1 strain is a biotin and methionine auxotroph (6, 7, 8, 10) and was used as a control in some experiments. The argB2 riboB2 strain was used to construct the double mutant strain by sexual crosses.

Aspergillus standard media (3) were used for the maintenance of the strains and for growth test and transformation experiments. A liquid defined medium was used to obtain mycelia, protein extracts, and microsomes and contained the following (in g/liter): KPO4H2, 13.2; (NH4)2SO4, 2; MgSO4·7H2O, 0.25; FeSO4·7H2O, 0.005; and CuSO4·5H2O, 0.001. The pH was adjusted to 7 with KOH, and glucose (0.3%) was added after autoclaving. Supplements were added when necessary.

Liquid cultures and mycelium harvesting were carried out as previously described (6, 10). To summarize, 108 conidiospores/ml were inoculated in minimal medium with required supplements, with glucose as the only carbon source, and grown for 18 h. The mycelium was harvested by filtration, washed, and transferred to liquid medium with phenylacetic acid (10 mM) as the only carbon source for 5 h in order to induce a PhAc catabolic pathway.

Preparation of microsomes and enzyme assay. Mycelium was resuspended (7 ml/g wet weight) in 100 mM potassium phosphate buffer, pH 7, and disrupted with a sonicator on ice (Branson digital sonifier 250; five pulses of 10 seconds each, with 60-second intervals and a 60% amplitude). Crude extracts were clarified after centrifugation at 22,000 x g for 15 min. The supernatant was tested for homogentisate dioxygenase activity (used as a control for induction) (7, 9). Microsomal pellets were recovered after centrifugation at 100,000 x g for 1 h and resuspended in 100 mM potassium phosphate buffer, pH 7.0. These extracts contained 2 to 4 mg/ml protein. NADPH-cytochrome P450 reductase and PhAc (and mono- and dihydroxyderivative) hydroxylating activities were assayed as described previously (10, 16). Phenylacetate and hydroxyderivatives were added at 1 mM (final concentration in the reaction mixture). Reaction mixtures were incubated at 37°C. Proteins in samples were precipitated with 0.1 volume of metaphosphoric acid (20%). After being centrifuged for 15 min, samples were filtered through syringe filters (PTFE; 0.45 µm), and 20 µl was analyzed by high-performance liquid chromatography (HPLC) with an Alliance system (Waters 2690), a Waters 996 diode array detector, and a Nucleosil C18 column with a 5-µm particle size (250 mm x 4.6 mm) (Supelco). The mobile phase was 50 mM NaH2PO4-acetonitrile (92.5:7.5 [vol/vol]), and the flow rate was 0.6 ml min–1. Detection was done at 210 nm. Occasionally, detection was done at 290 nm for specific determination of 2,5-OH-PhAc. Retention times were as follows: PhAc, 64,285 min; 2-OH-PhAc, 38,998 min; 3-OH-PhAc, 29,313 min; 4-OH-PhAc, 25,778 min; 2,5-diOH-PhAc, 11,705 min; and 3,4-diOH-PhAc, 15,570 min.

phacB gene disruption and construction of double mutant ({Delta}phacA {Delta}phacB) strain. For disruption of the phacB gene ({Delta}phacB), we used standard protocols (6, 10, 17). To summarize, a 4-kb linear DNA fragment (HindIII-XbaI) including the phacB gene and containing (internally) the argB+ gene in the BamHI-EcoRI sites of the phacB gene, which results in disruption of the phacB gene by the argB+ allele (argB+::phacB), was introduced by transformation into an arginine-deficient strain (argB2). Transformed clones were isolated in medium without arginine and selected for a single integration event in the phacB gene by means of Southern blotting. The resulting protein is truncated after proline 189. The handling of nucleic acids was undertaken according to standard procedures.

A double mutant strain was made by sexual crosses. The disrupted {Delta}phacB strain [{Delta}phacB (argB+::phacB) biA1 methG1] was crossed with an arginine- and riboflavin-deficient strain (argB2 riboB2), and the progeny argB+ (argB+::phacB riboB2) strain was crossed with the {Delta}phacA strain [{Delta}phacA (argB+::phacA) biA1 methG1] to obtain the double mutant strain ({Delta}phacA {Delta}phacB biA1).

GC-MS. For GC-MS analysis, 1 ml of reaction mixture was acidified with 20 µl of HCl (37%), and 500 µl of methyl-terbutyl ether was added. After being shaken vigorously by vortexing, samples were centrifuged for 15 min at 12,000 x g. The upper organic layer was collected and evaporated in a vacuum. Solid residue was resuspended in 25 µl of pyridine and 40 µl of BSTFA [N,O-bis(trimethylsilyl)trifluoroacetamide] as a derivatizing reagent. The mixture was incubated for 30 min at 80°C and was injected into a chromatograph (Varian CP-3800) with a Varian VF-5MS column (30 m x 0.25 mm [internal diameter] x 0.25-µm film thickness). The temperature program was 70°C (0 min), an increase to 200°C at 10°C/min, a hold at 200°C (7 min), an increase to 300°C at 100°C/min, and then a hold at 300°C (4 min). The carrier gas was helium at 1 ml/min, and the injector temperature was 250°C. The detector was a Satur 2200 ion trap (Varian), and the ion range was from 40 m/z to 550 m/z. Compounds were identified by comparing the obtained spectrum with the NIST/EPA/NIH mass spectrum library and a previously published spectrum of 2,4,5-trihydroxyphenylacetate (20).


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RESULTS AND DISCUSSION
 
Cloning, sequencing, and expression of the phacB gene. The phacB gene was isolated in a differential subtractive cDNA screen to isolate cDNAs induced by PhAc (6, 7, 8, 10). The phacB gene encodes a 517-amino-acid protein that contains the consensus sequence GXGXRXCXG (heme group binding site), which indicates it is a cytochrome P450. The gene has been classified as the first member of a new cytochrome P450 subfamily (CYP504B) by David Nelson (http://drnelson.utmem.edu/CytochromeP450.html). Genomic DNA sequencing showed that the phacB gene has three introns (71, 66, and 85 nucleotides). The sequence has GenBank accession number DQ217596 [GenBank] . A search through databases reveals similar genes in other fungi, with very high identities. The highest identity is found for protein 1397 (GenBank accession number XP 659001) from Aspergillus nidulans (99%), but without identity in the last 13 amino acids, which is probably the result of an error in the sequence of this protein. Moreover, we found a very high identity (84%) with a putative phenylacetate 2-hydroxylase (accession number EAL90209 [GenBank] ) from Aspergillus fumigatus. However, the protein from A. fumigatus has only 40% identity with the phenylacetate 2-hydroxylase (encoded by the phacA gene) from Aspergillus nidulans. For this reason, we think that it is probably the 3-hydroxyphenylacetate 6-hydroxylase from Aspergillus fumigatus. A protein (XP 748171) from A. fumigatus which displays 43% identity with the protein encoded by the phacB gene and 85% identity with the phacA-encoded protein should be the real phenylacetate 2-hydroxylase from A. fumigatus. Proteins from Gibberella zeae and Magnaporthe grisea (accession numbers XP 360809 and XP 75757, respectively) also share >60% identity.

There are also some proteins in plants (BAB03171 [GenBank] from Arabidopsis and BAA74465 [GenBank] from Glycyrrhiza) which share >25% identity throughout the full sequence and >45% identity of conserved residues.

The expression pattern of the phacB gene resembles that of some other genes involved in PhAc catabolism in A. nidulans (6, 7, 8, 10). It is strongly induced by PhAc and by all monohydroxyderivatives and dihydroxyderivatives, phenylalanine, and tyrosine. Glucose and acetate are not inducers (Fig. 2, lanes 2 and 11), and glucose (lane 3) repressed expression of the phacB gene.


Figure 2
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  		FIG. 2. Northern analysis of phacB gene expression. A. nidulans was grown in minimal medium with 0.3% glucose for 18 h and then transferred to medium with the following substrates: 1, PhAc; 2, glucose; 3, PhAc plus glucose; 4, 2-OH-PhAc; 5, 3-OH-PhAc; 6, 4-OH-PhAc; 7, phenylalanine; 8, tyrosine; 9, 2,5-diOH-PhAc; 10, 3,4-diOH-PhAc; and 11, acetate. All compounds were added at a concentration equivalent to 5 mM PhAc. Transferred cultures were incubated for 1 h at 37°C, and then RNAs were isolated.

Phenotype of {Delta}phacB strain. The growth of some Aspergillus mutant strains in minimal medium, with PhAc and phenylderivatives as the only carbon sources, is shown in Table 1. The {Delta}hmgA strain is not able to grow on PhAc, 2-hydroxy-, 3-hydroxy-, 4-hydroxy-, 2,5-dihydroxy-, and 3,4-dihydroxy-PhAc, which indicates that the catabolism of all these compounds is via 2,5-diOH-PhAc (homogentisic acid). The {Delta}phacA strain does not grow on PhAc (10), and the {Delta}phacB strain does not grow on 3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc. This shows that the phacB gene is involved in catabolism of 3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc.


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  		TABLE 1. Growth of some A. nidulans strains on PhAc and PhAc hydroxyderivatives

The {Delta}phacB strain was sexually crossed with an arginine- and riboflavin-deficient strain as the first step towards obtaining a double mutant strain ({Delta}phacA {Delta}phacB). No arginine-independent progeny were able to grow on 3-OH-PhAc, 4-OH-PhAc, or 3,4-diOH-PhAc. Auxotroph colonies requiring arginine were able to grow on these hydroxyderivatives. This shows that the argB+ allele disrupting the phacB gene is the only event which affects the use of 3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc.

Secretion of 2-OH-PhAc is reduced in {Delta}phacA and {Delta}phacB mutants. Aspergillus nidulans secretes 2-OH-PhAc into the culture medium when it is grown on PhAc as the carbon source (10). The accumulation of 2-OH-PhAc in culture supernatants of some mutants after the addition of PhAc is shown in Fig. 3. {Delta}phacA and {Delta}phacB strains showed a reduced accumulation of 2-OH-PhAc. The double mutant strain ({Delta}phacA {Delta}phacB) accumulated no 2-OH-PhAc. This shows that the phacA and phacB genes are involved in the conversion of PhAc to 2-OH-PhAc and that these two genes are solely responsible for 2-hydroxylation of PhAc. We know that the phacA gene encodes a phenylacetate 2-hydroxylase (10), and the growth of the {Delta}phacB mutant in hydroxyderivatives (Table 1) indicates that this gene is involved in the catabolism of 3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc. The decrease in 2-OH-PhAc secretion into the medium by the {Delta}phacB strain suggests that this mutant could be affected in ortho-hydroxylation of PhAc to produce 2-OH-PhAc. From this information, we can hypothesize that the phacB gene encodes a 3-hydroxyphenylacetate 6-hydroxylase which produces 2,5-diOH-PhAc from 3-OH-PhAc but which is also able to catalyze the 2-hydroxylation of PhAc to 2-OH-PhAc, probably due to structural analogy between PhAc and 3-hydroxy-PhAc and the lack of specificity of the enzyme encoded by the phacB gene (Fig. 1 and 3; see below).


Figure 3
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  		FIG. 3. Secretion of 2-hydroxyphenylacetic acid by A. nidulans. A. nidulans was grown in minimal medium with 0.3% glucose for 18 h and then transferred to medium with 5 mM phenylacetate. At the appropriate moment, 1 ml of medium was harvested, filtered, and analyzed by HPLC. Wild-type ({blacksquare}), {Delta}phacA ({blacklozenge}), {Delta}phacB ({blacktriangleup}), and {Delta}phacA {Delta}phacB double mutant (•) strains were examined.

Phenylacetate is a precursor in penicillin biosynthesis. A lack of PhAc hydroxylation should increase penicillin production and decrease PhAc consumption. For this reason, the industry has been looking for a non-PhAc-hydroxylating strain of Penicillium chrysogenum (the industrial producer of penicillin) for many years. It has been shown that phacA mutant strains of Penicillium and A. nidulans increase penicillin production (10, 14). However, {Delta}phacB and double mutant ({Delta}phacA {Delta}phacB) strains of A. nidulans did not increase penicillin production compared to that of the {Delta}phacA strain (data not shown), which also suggests a minor role for the phacB gene in hydroxylation of PhAc in P. chrysogenum.

In vitro hydroxylation of PhAc and hydroxyderivatives. To clearly define the roles of the phacA and phacB genes in hydroxylation of PhAc and its hydroxyderivatives, we assayed the hydroxylating capacity of some Aspergillus nidulans strains in vitro by using microsomes. Hydroxyderivatives, the products of the reactions, were analyzed by HPLC. When PhAc was the substrate, microsomes from the wild-type strain were able to catalyze the production of 2-OH-PhAc, and microsomes from the {Delta}phacA strain catalyzed this hydroxylation to a lesser extent (10) (Fig. 4, top panel). Microsomes from the {Delta}phacB strain catalyzed 2-hydroxylation of PhAc to the same extent as the wild-type strain. Double mutant ({Delta}phacA {Delta}phacB) microsomes were not able to produce any 2-OH-PhAc, which again indicates a minor role of the phacB gene in 2-hydroxylation of PhAc. However, this is the second described gene to encode a protein with PhAc 2-hydroxylase activity.


Figure 4
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  		FIG. 4. In vitro synthesis of 2-hydroxyphenylacetate from phenylacetate (top) and of 2,5-dihydroxyphenylacetate from 3-hydroxyphenylacetate (bottom) by A. nidulans microsomal fractions from wild-type ({blacksquare}), {Delta}phacA ({blacklozenge}), {Delta}phacB ({blacktriangleup}), and {Delta}phacA {Delta}phacB double mutant (•) strains.

When the substrate was 3-OH-PhAc (Fig. 4, bottom panel), microsomes from the wild-type and {Delta}phacA strains generated 2,5-diOH-PhAc. Microsomes of the {Delta}phacB strain produced 2,5-diOH-PhAc to a lesser extent than did those of the wild-type strain, which demonstrates the major role of the phacB gene in hydroxylation of 3-OH-PhAc. The double mutant microsomes ({Delta}phacA {Delta}phacB) produced no 2,5-diOH-PhAc, which also indicates that the phacA gene has a minor role in 6-hydroxylation of 3-OH-PhAc, perhaps due to the structural analogy of 3-OH-PhAc and PhAc. NADPH-cytochrome P450 oxidoreductase was assayed as a control (10, 16) in the wild-type and mutant strains, and all of them showed similar levels of activity with ferricyanide (680 to 810 nmol/min·mg in all assays) and cytochrome c (250 to 290 nmol/min·mg in all assays) as substrates.

Figure 1 shows the hydroxylation activities of PhAc and 3-hydroxy-PhAc mediated by the phacA and phacB genes.

3-Hydroxyphenylacetate 6-hydroxylases from bacteria (Flavobacterium) and fungi (Trichosporon) have been described previously (1, 11, 18, 19), and recently a gene from Pseudomonas putida encoding a two-component enzyme with 3-hydroxyphenylacetate 6-hydroxylase activity was cloned (2). However, Aspergillus nidulans is a eukaryotic microorganism which, unlike bacteria, uses cytochromes P450 to hydroxylate the aromatic rings of PhAc and its hydroxyderivatives. The phacA and phacB genes are examples of genes encoding these fungal hydroxylases.

An A. nidulans strain which is deficient in homogentisate dioxygenase activity ({Delta}hmgA) is not able to grow on 4-hydroxy- and 3,4-dihydroxy-PhAc as the only carbon source (Table 1), indicating that the catabolism of these compounds is through the homogentisic acid pathway. It is easy to explain the conversion of 3-OH-PhAc to 2,5-diOH-PhAc (Fig. 1) but more difficult to explain the conversion of 4-OH-PhAc and 3,4-diOH-PhAc to a compound similar to homogentisic acid (2,5-diOH-PhAc) which is utilized by homogentisate dioxygenase as a substrate. Anderson and Dagley (1) proposed a catabolic pathway in Trichosporon cutaneum which metabolizes 4-OH-PhAc and 3,4-diOH-PhAc to a trihydroxyderivative (2,4,5-trihydroxy-PhAc), which is converted by homogentisate dioxygenase (or a similar enzyme) to oxalacetylacetoacetate (Fig. 1). The 3-OH-PhAc 6-hydroxylase enzyme from Flavobacterium is able to convert 3,4-diOH-PhAc to 2,4,5-triOH-PhAc (18, 19), and there is no indication of substrate use for the Pseudomonas enzyme (2). For this reason, we also assayed the formation of 2,4,5-trihydroxyphenylacetate from 3,4-dihydroxyphenylacetate and 4-hydroxy-PhAc. We used GC-MS to analyze these reactions because the absorption spectra of 2,5-diOH-PhAc and 2,4,5-triOH-PhAc and their retention times (in our HPLC system) are very similar. Microsomes of the wild-type strain, the {Delta}phacA mutant, the {Delta}phacB mutant, and the {Delta}phacA {Delta}phacB double mutant catalyze the formation of 3,4-dihydroxyphenylacetate from 4-hydroxyphenylacetate (data not shown). Microsomes of the wild-type strain and the {Delta}phacA mutant are able to catalyze the formation of 2,4,5-trihydroxyphenylacetate from 3,4-dihydroxy-PhAc (Fig. 5), but microsomes of the {Delta}phacB mutant strain cannot catalyze the formation of 2,4,5-trihydroxyphenylacetate (Fig. 5). The true nature of this trihydroxyderivative was confirmed by gas chromatography-mass spectrometry (Fig. 6), clearly indicating that the phacB gene is also responsible for the synthesis of 2,4,5-trihydroxyphenylacetate from 3,4-dihydroxyphenylacetate. This trihydroxyderivative has also been found in the urines of patients with Parkinson's disease treated with L-DOPA (3,4-dihydroxyphenylalanine) (20).


Figure 5
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  		FIG. 5. Gas chromatography analysis of in vitro synthesis of 2,4,5-trihydroxyphenylacetate (retention time, 17.770 min) from 3,4-dihydroxyphenylacetate (retention time, 14.775 min).


Figure 6
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  		FIG. 6. (A) Mass spectrum analysis of the 2,4,5-trihydroxyphenylacetate shown in Fig. 5 (retention time, 17.770 min). (B) Mass spectrum of 2,4,5-trihydroxyphenylacetate obtained from NIST/EPA/NIH mass spectrum library.

We should also note that homogentisate dioxygenase (hmgA) from A. nidulans is the enzyme responsible for opening the aromatic ring of 2,4,5-triOH-PhAc. The lack of growth of the {Delta}hmgA strain on 4-OH-PhAc and 3,4-diOH-PhAc indicates that homogentisate dioxygenase from A. nidulans is the enzyme responsible for catabolizing 2,4,5-trihydroxy-PhAc and proves that Aspergillus nidulans, unlike other microorganisms, is able to catabolize PhAc and its mono- and dihydroxyderivatives via the same pathway.

Also, phacB could be important in plant metabolism, as homogentisic acid is a precursor of some photosynthetic pigments (plastoquinone and tocopherols) (12) and because there are some genes with identity to phacB in plant genomes.


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ACKNOWLEDGMENTS
 
This investigation was made possible by a grant from the Ministry of Science and Technology (BCM 2003-01592), by a grant from the Castilla and León Regional Government (LE06/04), and by a contract with Antibioticos SA (Spain). F.F.S. was the recipient of a fellowship from grant BCM 2003-01592.

We thank David Nelson for the standardized cytochrome P450 designation of phacB, J. Carlos García González for his advice with HPLC and GC-MS analysis, and Richard Prowse and Mark Hayward for proofreading the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Biología Molecular, Genómica y Proteómica, Universidad de León, Campus de Vegazana, s/n 24071 León, Spain. Phone: 34-987-291975. Fax: 34-987-291282. E-mail: jose.fernandez.canon{at}unileon.es. Back

{triangledown} Published ahead of print on 22 December 2006. Back


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REFERENCES
 
    1
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Eukaryotic Cell, March 2007, p. 514-520, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00226-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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