Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities

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).

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.

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.

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 (▪), ΔphacA (⧫), ΔphacB (▴), and ΔphacA ΔphacB double mutant (•) strains were examined.

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 (▪), ΔphacA (⧫), ΔphacB (▴), and ΔphacA ΔphacB double mutant (•) strains.

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).

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.