Purification and properties of 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase involved in microbial degradation of carbazole.
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 degrees C. The calculated K'(m) for HOPDA was 4.6 microM, the V(max) was 3.3 micromol min(-1), and the K(s) was 70.0 microM. This corresponds to a maximum specific turnover rate of 1300 HOPDAs(-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.
Riddle
Robert R
RR
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
Gibbs
Phillip R
PR
Willson
Richard C
RC
Benedik
Michael J
MJ
D002227:Carbazoles / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
D002227:Carbazoles; C041514:carbazole; C476804:2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid hydrolase; D006867:Hydrolases