title: Gene expression and biochemical characterization of Azotobacter vinelandii cyclophilins and Protein Interaction Studies of the cytoplasmic isoform with dnaK and lpxH.
abstract: The soil nitrogen-fixing bacterium Azotobacter vinelandii possesses two cyclophilins, comprising putative cytoplasmic and periplasmic isoforms, designated as AvPPIB and AvPPIA, respectively. Both recombinant cyclophilins have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides has been characterized. The substrate specificity of both cyclophilins is typical for bacterial cyclophilins, with Suc-Ala-Ala-Pro-Phe-pNA being the most rapidly catalyzed substrate. The cytoplasmic cyclophilin also displays a chaperone function in the citrate synthase thermal aggregation assay. Using real-time quantitative RT-PCR, we demonstrate that AvppiB is expressed under various physiological and growth conditions, mainly upregulated by acetate and downregulated by the stationary growth state, while AvppiA shows a tendency for downregulation under the tested conditions. Further, we identified chaperone protein dnaK and UDP-2, 3-diacylglucosamine hydrolase lpxH as probable interacting partners of AvPPIB and we demonstrate their physical interaction by coexpression studies. An increase in AvPPIB PPIase activity in the presence of AvdnaK and a decrease in the presence of AvlpxH further confirms each interaction. However, the PPIase activity does not seem to be essential for those interactions since AvPPIB active site mutants still interact with dnaK and lpxH, while their minor PPIase activity cannot be modulated by the interaction.
authors: Department of Agricultural Biotechnology, Agricultural University of Athens, Greece.
authors: Maria
authors: M
authors: Dimou
authors: Anastasia
authors: A
authors: Venieraki
authors: Georgios
authors: G
authors: Liakopoulos
authors: Evangelia D
authors: ED
authors: Kouri
authors: Anastasia
authors: A
authors: Tampakaki
authors: Panagiotis
authors: P
authors: Katinakis
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021983:Cyclophilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D007527:Isoenzymes; D018832:Molecular Chaperones; D009842:Oligopeptides; D011994:Recombinant Proteins; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; C464799:UDP-2,3-diacylglucosamine pyrophosphatase; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase