Characterization of protocatechuate 4,5-dioxygenase induced from p-hydroxybenzoate-cultured Pseudomonas sp. K82.
Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, p-hydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 (alpha subunit and beta subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer (alpha1beta1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 degrees C. PCR amplification of these two subunits of PCD4,5 revealed that the alpha subunit and beta subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.
Yun
Sung-Ho
SH
Proteome Analysis Team, Korea Basic Science Institute, Daejeon, 305-806, Korea.
Yun
Chi-Young
CY
Kim
Seung Il
SI
D000595:Amino Acid Sequence; D020604:Comamonas testosteroni / Q000235:genetics; D019281:Dimerization; D049308:Dioxygenases; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism*; D010226:Parabens / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D062385:Hydroxybenzoates; D010226:Parabens; D021122:Protein Subunits; C009091:protocatechuic acid; D010105:Oxygenases; D049308:Dioxygenases; C022476:protocatechuate 4,5-dioxygenase; C038193:4-hydroxybenzoic acid