PURIFICATION AND CHARACTERIZATION OF THE 4-HYDROXYPHENYLACETIC ACID-3-HYDROXYLASE FROM PSEUDOMONAS-PUTIDA U

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 degrees C. Under these conditions, the K-m values calculated fo r 4-hydroxyphenylacetic acid, NADH and FAD were 38, 41 and 4 mu M resp ectively. The native enzyme (M-r 65000) had two identical subunits in an alpha(2) oligomeric structure and required the addition of FAD, so it was classified as an external flavoprotein monooxygenase. 4-Hydroxy phenylacetic acid-3-hydroxylase showed a broad substrate range. It was specifically induced by 4-hydroxyphenylacetic acid, although phenylac etic acid and some phenyl-alkanoic acids also induced enzymatic activi ty to a lesser extent. 4-Hydroxyphenylacetic acid-3-hydroxylase induct ion 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.