TY - JOUR
T1 - Purification and properties of 2-hydroxy-6-oxo-6(2′-aminophenyl)hexa-2,4-dienoic acid hydrolase involved in microbial degradation of carbazole
AU - Riddle, Robert R.
AU - Gibbs, Phillip R.
AU - Willson, Richard C.
AU - Benedik, Michael J.
N1 - Funding Information:
We thank Phil Fedorak and Lisa Geig for supplying the strain LD2. We gratefully acknowledge Enchira Corporation (formerly Energy Biosystems), the Energy Lab and Environmental Institute of the University of Houston, the Robert A. Welch Foundation, and Texas Advanced Research Program for funding this research.
PY - 2003/3/1
Y1 - 2003/3/1
N2 - 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 His6-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 Vmax was 3.3 μmol min-1, and the Ks 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.
AB - 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 His6-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 Vmax was 3.3 μmol min-1, and the Ks 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.
KW - Carbazole
KW - HOPDA
KW - Hydrolase
KW - Meta-Cleavage enzyme
KW - Pseudomonas
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U2 - 10.1016/S1046-5928(02)00676-9
DO - 10.1016/S1046-5928(02)00676-9
M3 - Article
C2 - 12651123
AN - SCOPUS:0345381843
SN - 1046-5928
VL - 28
SP - 182
EP - 189
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -