Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from Rhodococcus sp. R04, showing their distinct stability at various temperature.
The genes of two 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC1 and BphC2) were obtained from the gene library of Rhodococcus sp. R04. The enzymes have been purified to apparent electrophoretic homogeneity from the cell extracts of the recombinant harboring bphC1 and bphC2. Both BphC1 and BphC2 were hexamers, consisting of six subunits of 35 and 33 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzymes had similar optimal pH (pH 9.0), but different temperatures for their maximum activity (30 degrees C for BphC1, 80 degrees C for BphC2). In addition, they exhibited distinct stability at various temperatures. The enzymes could cleave a wide range of catechols, with 2,3-dihydroxybiphenyl being the optimum substrate for BphC1 and BphC2. BphC1 was inhibited by 2,3-dihydroxybiphenyl, catechol and 3-chlorocatechol, whereas BphC2 showed strong substrate inhibition for all the given substrates. BphC2 exhibited a half-life of 15 min at 80 degrees C and 50 min at 70 degrees C, making it the most thermostable extradiol dioxygenase studied in mesophilic bacteria. After disruption of bphC1 and bphC2 genes, R04DeltaC1 (bphC1 mutant) delayed the time of their completely eliminating biphenyl another 15 h compared with its parent strain R04, but R04DeltaC2 (bphC2 mutant) lost the ability to grow on biphenyl, suggesting that BphC1 plays an assistant role in the degrading of biphenyl by strain R04, while BphC2 is essential for the growth of strain R04 on biphenyl.
Yang
Xiuqing
X
Institute of Biotechnology, Shanxi University, Taiyuan, China.
Xie
Fuhong
F
Zhang
Guoqing
G
Shi
Yawei
Y
Qian
Shijun
S
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011994:Recombinant Proteins; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase; C439587:extradiol dioxygenase