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Biochemical characterization of two Azotobacter vinelandii FKBPs and analysis of their interaction with the small subunit of carbamoyl phosphate synthetase

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Abstract

FK-506 binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase superfamily (PPIases, EC: 5.2.1.8) which catalyzes the interconversion of peptidyl-prolyl bonds while they can also act on polypeptides, as folding helper enzymes. Here, we biochemically characterize two recombinant FKBPs, AvfkbA1 and AvfkbA2, from the soil nitrogen-fixing bacterium Azotobacter vinelandii and show that both possess PPIase activity while AvfkbA2 possesses chaperone activity as well. Further, we demonstrate their physical interaction with AvcarA, the small subunit of carbamoyl phosphate synthetase. Using RT-qPCR, we show that AvfkbA1 and AvfkbA2 are co-expressed with AvcarA under the same growth conditions. A decrease in AvfkbA1 or AvfkbA2 PPIase activity, in the presence of AvcarA, further confirms each interaction. However, PPIase activity does not seem to be essential for these interactions since PPIase active site mutations of both FKBPs do not abolish the AvcarA binding. The P358 residue of AvcarA, possibly retaining a cis configuration, is critical only for the interaction with AvfkbA1. The presence of either of the two FKBPs did not influence the measured glutamine hydrolyzing activity of AvcarA. Taken together, these data indicate that although the two FKBPs have a common biological substrate they probably have differing physiological roles.

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Abbreviations

FKBPs:

FK-506 binding proteins

PPIases:

Peptidyl-prolyl cis/trans isomerase

RT-qPCR:

Quantitative real time polymerase chain reaction

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Authors and Affiliations

  1. Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Votanikos, 11855, Athens, Greece

    Maria Dimou, Chrysoula Zografou, Anastasia Venieraki & Panagiotis Katinakis

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  1. Maria Dimou
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  2. Chrysoula Zografou
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Correspondence to Panagiotis Katinakis.

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Dimou, M., Zografou, C., Venieraki, A. et al. Biochemical characterization of two Azotobacter vinelandii FKBPs and analysis of their interaction with the small subunit of carbamoyl phosphate synthetase. Mol Biol Rep 39, 10003–10012 (2012). https://doi.org/10.1007/s11033-012-1869-4

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