Purification and characterization of the 4-hydroxyphenylacetic acid-3-hydroxylase from Pseudomonas putida U |
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Authors: | Alberto Fernández-Medarde José M Luengo |
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Institution: | Departamento de Bioqu??mica y Biolog??a Molecular, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain |
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Abstract: | 4-Hydroxyphenylacetic acid-3-hydroxylase from Pseudomonas putida U was purified to homogeneity (96-fold) from bacterial cultures grown in a chemically defined medium containing 4-hydroxyphenylacetic acid as the sole carbon source. The maximal rate of catalysis occurred at pH 7.5 and 40°C. Under these conditions, the Km values calculated for 4-hydroxyphenylacetic acid, NADH and FAD were 38, 41 and 4 μM respectively. The native enzyme (Mr 65 000) had two identical subunits in an α2 oligomeric structure and required the addition of FAD, so it was classified as an external flavoprotein monooxygenase. 4-Hydroxyphenylacetic acid-3-hydroxylase showed a broad substrate range. It was specifically induced by 4-hydroxyphenylacetic acid, although phenylacetic acid and some phenyl-alkanoic acids also induced enzymatic activity to a lesser extent. 4-Hydroxyphenylacetic acid-3-hydroxylase induction and 4-hydroxyphenylacetic acid consumption were unaffected by the presence of glucose, suggesting that the uptake and hydroxylation of 4-hydroxyphenylacetic acid are not under carbon catabolite repression. |
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Keywords: | 4-Hydroxyphenylacetic acid Hydroxylase Aromatics |
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