Influence of the histidine tail on the structure and activity of recombinant chlorocatechol 1,2-dioxygenase.
We present two efficient expression systems for the chlorocatechol 1, 2-dioxygenase (CCD) from Pseudomonas putida. In the first, CCD (encoded by the clcA gene) was expressed in the pETCLCA vector with the addition of an N-terminal histidine tail. After purification, the enzyme (CCD 6xHis) was proteolytically cleaved with thrombin to remove the His tail. The CD spectra of the cleaved and uncleaved enzymes present only minor differences, indicative of correct protein folding. However, the activity of CCD 6xHis, over a wide range of pH, was typically five times lower. This may be the result of steric hindrance caused by the histidine tail. These data are consistent with results obtained using an alternative construct employing a vector which produces a protein product devoid of the His tail. These results suggest that the His tail may induce subtle effects close to the active site which compromise the recovery of full biological activity.
Araújo
A P
AP
Departamento Física e Informática, Instituto de Física de São Carlos/USP, São Carlos, São Paulo, Brazil.
Oliva
G
G
Henrique-Silva
F
F
Garratt
R C
RC
Cáceres
O
O
Beltramini
L M
LM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D049308:Dioxygenases; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016958:Pseudomonas putida / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D010105:Oxygenases; D049308:Dioxygenases; C044173:chlorocatechol 1,2-dioxygenase; D013917:Thrombin