Eukaryotic Cell
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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

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).

* 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.

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

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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