title: Crystal structure of a histidine-tagged serine hydrolase involved in the carbazole degradation (CarC enzyme).
abstract: 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=130.3A, c=84.5A in the hexagonal setting, and the crystal structure of ht-CarC(J3) was determined at 1.86A 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.
authors.affiliation: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Habe
authors.forename: Kenichi
authors.initials: K
authors.lastname: Morii
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Jeong-Won
authors.initials: JW
authors.lastname: Nam
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ayabe
authors.forename: Takako
authors.initials: T
authors.lastname: Yoshida
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Wakagi
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Yamane
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nojiri
authors.forename: Toshio
authors.initials: T
authors.lastname: Omori
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D002227:Carbazoles; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002227:Carbazoles; D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D006639:Histidine; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; D012697:Serine Endopeptidases