Open AccessPurification and characterization of the 4‐hydroxyphenylacetic acid‐3‐hydroxylase from Pseudomonas putida U
Author(s) -
FernándezMedarde Alberto,
Luengo José M
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb12751.x
Subject(s) - pseudomonas putida , phenylacetic acid , hydroxylation , chemistry , catabolite repression , enzyme , monooxygenase , flavoprotein , biochemistry , substrate (aquarium) , biology , cytochrome p450 , mutant , ecology , gene
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 K m values calculated for 4‐hydroxyphenylacetic acid, NADH and FAD were 38, 41 and 4 μM respectively. The native enzyme ( M r 65 000) had two identical subunits in an α 2 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.