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PMID:10913088
Citation |
Howell, ML, Alsabbagh, E, Ma, JF, Ochsner, UA, Klotz, MG, Beveridge, TJ, Blumenthal, KM, Niederhoffer, EC, Morris, RE, Needham, D, Dean, GE, Wani, MA and Hassett, DJ (2000) AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. J. Bacteriol. 182:4545-56 |
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Abstract |
In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification. |
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Keywords |
Amino Acid Sequence; Ankyrin Repeat; Ankyrins/chemistry; Ankyrins/genetics; Ankyrins/metabolism; Catalase/genetics; Catalase/metabolism; Cell Membrane/metabolism; Cell Membrane/ultrastructure; Chromosome Mapping; Cloning, Molecular; Hydrogen Peroxide/pharmacology; Molecular Sequence Data; Periplasmic Proteins; Plasmids; Protein Conformation; Pseudomonas aeruginosa/drug effects; Pseudomonas aeruginosa/genetics; Pseudomonas aeruginosa/metabolism; Pseudomonas aeruginosa/ultrastructure; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/metabolism; Sequence Alignment; Sequence Homology, Amino Acid |
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Significance
Annotations
Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
---|---|---|---|---|---|---|---|---|
GO:0043580: periplasmic space organization |
ECO:0000315: |
P |
Fig. 6b |
complete | ||||
GO:0043580: periplasmic space organization |
ECO:0000315: |
P |
Fig. 6 |
complete | ||||
GO:0010628: positive regulation of gene expression |
ECO:0000315: |
P |
Fig. 8 |
complete | ||||
See also
References
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