Identification of a novel gentisate 1,2-dioxygenase from Silicibacter pomeroyi.
A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K (m) and K (cat)/K (m) for gentisate were 12 muM and 653 x 10(4) M(-1 )s(-1); the pI was 4.6-4.8. It was optimally active at 40 degrees C and pH 8.0.
Liu
Dongqi
D
College of Environmental Sciences, Peking University, Beijing, 100871, P.R. China. liudongqi@iee.pku.edu.cn
Zhu
Tingting
T
Fan
Li
L
Quan
Junming
J
Guo
Hongchun
H
Ni
Jinren
J
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D041902:Rhodobacteraceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase