Purification and properties of 2-hydroxy-6-oxo-6-(2 ′-aminophenyl)hexa-2,4-dienoic acid hydrolase involved in microbial degradation of carbazole

Abstract

Hydrolysis following meta-ring cleavage by a dioxygenase is a well-known step in aromatic compound metabolism. The 2-hydroxy-6-oxo-6-(2 ′-aminophenyl)hexa-2,4-dienoic acid hydrolase from Pseudomonas LD2 is a new member of the small group of characterized aromatic hydrolases that catalyze the cleavage of C–C bonds. In this study, the His 6-tagged 2-hydroxy-6-oxo-6-(2 ′-aminophenyl)hexa-2,4-dienoic acid (HOPDA) hydrolase was purified from a recombinant Escherichia coli strain utilizing immobilized metal affinity chromatography. 2-Hydroxy-6-oxo-6-(2 ′-aminophenyl)hexa-2,4-dienoic acid hydrolase is a colorless homodimer with no cofactor requirement. The enzyme actively converted HOPDA into benzoic acid and 2-hydroxypenta-2,4-dienoic acid. The enzyme exhibited activity between pH 6.5 and 10.5 with a maximum activity at pH 7.0. The optimum temperature at pH 7.0 was 60 °C. The calculated K ′ m for HOPDA was 4.6 μM, the V max was 3.3 μmol min −1, and the K s was 70.0 μM. This corresponds to a maximum specific turnover rate of 1300 HOPDA s −1 dimer −1 . The deduced amino acid sequence of CarC showed 30.3, 31.3, and 31.8% identity with TodF ( P. putida F1), XylF ( P. putida), and DmpD ( Pseudomonas sp. CF600), respectively, which are meta-cleavage compound hydrolases from other Pseudomonads. The amino acid sequence Gly–X–Ser–X–Gly, which is highly conserved in these hydrolases, is also found in CarC. Lysates from a strain expressing enzyme in which the putative active site serine is mutated to alanine showed a significant reduction in activity.