A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli.
Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of alpha- and beta-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K(m)=0.77 microM) than for 2-aminophenol (K(m)=0.89 microM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the alpha- and beta-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the beta- and alpha-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.
Wu
Jian-Feng
JF
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Sun
Cui-Wei
CW
Jiang
Cheng-Ying
CY
Liu
Zhi-Pei
ZP
Liu
Shuang-Jiang
SJ
D000595:Amino Acid Sequence; D000627:Aminophenols / Q000378:metabolism; D001673:Biodegradation, Environmental; D002733:Chlorophenols / Q000378:metabolism; D003001:Cloning, Molecular; D020585:Comamonas / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003360:Cosmids; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009578:Nitrobenzenes / Q000378:metabolism*; D016366:Open Reading Frames; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014871:Water Microbiology
D000627:Aminophenols; D002733:Chlorophenols; D004269:DNA, Bacterial; D062385:Hydroxybenzoates; D009578:Nitrobenzenes; D021122:Protein Subunits; D011994:Recombinant Proteins; C027667:2-aminophenol; C099673:2-amino-5-chlorophenol; C009091:protocatechuic acid; C010407:4-chloronitrobenzene; C106307:2-aminophenol 1,6-dioxygenase; D049308:Dioxygenases