title: 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).
authors.affiliation: Instituto de Biología Molecular, Genómica y Proteómica, Universidad de León, Campus de Vegazana, s/n 24071 León, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ferrer-Sevillano
authors.forename: José M
authors.initials: JM
authors.lastname: Fernández-Cañón
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000737:chemistry / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D050560:Homogentisate 1,2-Dioxygenase / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D015394:Molecular Structure; D010648:Phenylacetates / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: C071365:3-hydroxyphenylacetate 6-hydroxylase; D010648:Phenylacetates; D015102:3,4-Dihydroxyphenylacetic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050560:Homogentisate 1,2-Dioxygenase