Novel genes encoding 2-aminophenol 1,6-dioxygenase from Pseudomonas species AP-3 growing on 2-aminophenol and catalytic properties of the purified enzyme.
2-Aminophenol 1,6-dioxygenase was purified from the cell extracts of Pseudomonas sp. AP-3 grown on 2-aminophenol. The product from 2-aminophenol by catalysis of the purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas chromatographic and mass spectrometric analyses. The molecular mass of the native enzyme was 140 kDa based on gel filtration. It was dissociated into molecular mass subunits of 32 (alpha-subunit) and 40 kDa (beta-subunit) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase was a heterotetramer of alpha2beta2. The genes coding for the alpha- and beta-subunits of the enzyme were cloned and sequenced. Open reading frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively. The amino acid sequences predicted from the open reading frames of amnA and amnB corresponded to the NH2-terminal amino acid sequences of the alpha-subunit (AmnA) and beta-subunit (AmnB), respectively. The deduced amino acid sequences of AmnB showed identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are homoprotocatechuate 2,3-dioxygenases from Escherichia coli W and C, respectively, belonging to class III in the extradiol dioxygenases. On the other hand, AmnA had identity (23.3%) with only AmnB among the enzymes examined.
Takenaka
S
S
Division of Science of Biological Resources, Graduate School of Science and Technology, Kobe University, Rokko, Kobe 657, Japan.
Murakami
S
S
Shinke
R
R
Hatakeyama
K
K
Yukawa
H
H
Aoki
K
K
D000595:Amino Acid Sequence; D000627:Aminophenols / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D049308:Dioxygenases; D008401:Gas Chromatography-Mass Spectrometry; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010105:Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000627:Aminophenols; D046911:Macromolecular Substances; C027667:2-aminophenol; C106307:2-aminophenol 1,6-dioxygenase; D010105:Oxygenases; D049308:Dioxygenases