Crystal structure of a histidine-tagged serine hydrolase involved in the carbazole degradation (CarC enzyme)

2-Hydroxy-6-oxo-6-(2 ′-aminophenyl)-hexa-2,4-dienoate hydrolases (CarC enzymes) from two carbazole-degrading bacteria were purified using recombinant Escherichia coli strains with the histidine (His)-tagged purification system. The His-tagged CarC (ht-CarC) enzymes from Pseudomonas resinovorans stra...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2003-04, Vol.303 (2), p.631-639
Main Authors: Habe, Hiroshi, Morii, Kenichi, Fushinobu, Shinya, Nam, Jeong-Won, Ayabe, Yuko, Yoshida, Takako, Wakagi, Takayoshi, Yamane, Hisakazu, Nojiri, Hideaki, Omori, Toshio
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacteria - enzymology
Carbazole degradation
Carbazoles
Cloning, Molecular
Crystallography, X-Ray
DNA Primers
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Histidine
Histidine-tagged protein
Janthinobacterium
Kinetics
meta-cleavage product hydrolase
Models, Molecular
Molecular Sequence Data
Oxidoreductases - chemistry
Oxidoreductases - metabolism
Polymerase Chain Reaction
Protein Conformation
Protein Subunits - chemistry
Pseudomonas
Pseudomonas fluorescens
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Rhodococcus
Sequence Alignment
Sequence Homology, Amino Acid
Sequence Tagged Sites
Serine Endopeptidases - chemistry
Serine Endopeptidases - metabolism
X-ray crystallography
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Summary:2-Hydroxy-6-oxo-6-(2 ′-aminophenyl)-hexa-2,4-dienoate hydrolases (CarC enzymes) from two carbazole-degrading bacteria were purified using recombinant Escherichia coli strains with the histidine (His)-tagged purification system. The His-tagged CarC (ht-CarC) enzymes from Pseudomonas resinovorans strain CA10 (ht-CarC CA10) and Janthinobacterium sp. strain J3 (ht-CarC J3) exhibited hydrolase activity toward 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate as the purified native CarC CA10 did. ht-CarC J3 was crystallized in the space group I422 with cell dimensions of a= b=130.3 Å, c=84.5 Å in the hexagonal setting, and the crystal structure of ht-CarC J3 was determined at 1.86 Å resolution. The final refined model of ht-CarC J3 yields an R-factor of 21.6%, although the electron-density corresponding to Ile146 to Asn155 was ambiguous in the final model. We compared the known structures of BphD from Rhodococcus sp. strain RHA1 and CumD from Pseudomonas fluorescens strain IP01. The backbone conformation of ht-CarC J3 was better superimposed with CumD than with BphD RHA1. The side-chain directions of Arg185 and Trp262 residues in the substrate binding pockets of these enzymes were different among these proteins, suggesting that these residues may take a conformational change during the catalytic cycles.
ISSN:0006-291X
1090-2104
DOI:10.1016/S0006-291X(03)00375-9