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.
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
VL - 28
SP - 182
EP - 189
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -