Enhanced detection and characterization of protocatechuate 3,4-dioxygenase in Acinetobacter lwoffii K24 by proteomics using a column separation.
Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.
Kahng
Hyung Yeel
HY
Research Institute for Basic Sciences, Cheju National University, Jeju, Republic of Korea.
Cho
Kun
K
Song
Seung Yual
SY
Kim
Soo Jung
SJ
Leem
Sun Hee
SH
Kim
Seung Il
SI
D000150:Acinetobacter / Q000201:enzymology*; D000272:Adipates / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002845:Chromatography; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011520:Protocatechuate-3,4-Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D000272:Adipates; D001426:Bacterial Proteins; C027316:3-oxoadipic acid; D011520:Protocatechuate-3,4-Dioxygenase