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The cytoplasmic cyclophilin from Azotobacter vinelandii interacts with phosphate acetyltransferase isoforms enhancing their in vitro activity

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Abstract

Cyclophilins belong to the peptidyl-prolyl cis/trans isomerase family of enzymes (EC 5.2.1.8), which accelerate protein folding by catalysing the cis/trans isomerisation of proline imidic peptide bonds. In the present study, by a combination of bioinformatics methods, we identify phosphate acetyltransferase isoforms, AvPTA-1 and AvPTA-2, as potential interacting partners of AvPPIB, the cytoplasmic cyclophilin from Azotobacter vinelandii, and demonstrate their physical interaction by co-expression studies. A decrease in AvPPIB PPIase activity, in the presence of AvPTA-1 or AvPTA-2, further confirms each interaction. Phosphate acetyltransferases (EC 2.3.1.8) catalyse the reversible transfer of the acetyl group from acetyl-P to CoA, forming acetyl-CoA and inorganic phosphate. We examined the effect of AvPPIB on the enzymatic activity of both phosphate acetyltransferase isoforms, and noticed an enhancement of the activity, as well as an alteration of the K m of each isoform, for the reaction substrates, indicating a possible function of AvPPIB in phosphate acetyltransferase activity modulation. Although PPIase activity seems not to be essential for these interactions, since AvPPIBF99A active site mutant still interacts with both isoforms, it is responsible for the observed phosphate acetyltransferase activity enhancement as AvPPIBF99A enhanced to a significantly lower extent the phosphate acetyltransferase activity of both isoforms compared with AvPPIB.

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Abbreviations

CoA:

Coenzyme A

P:

Phosphate

PPIase:

Peptidyl-prolyl cis/trans isomerase

RT-qPCR:

Real-time quantitative polymerase chain reaction

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Correspondence to Panagiotis Katinakis.

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Fig. S1

Alignment of Methanosarcina thermophila pta (AAA72041) with Azotobacter vinelandii AvPTA-1 (ACO79604) and AvPTA-2 (ACO80246). Multiple sequence alignment was performed using ClustalW (Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500). Black boxes indicate identical amino acids while grey boxes indicate similar. Conserved prolines corresponding to cis peptide Gly295-Pro296 (Methanosarcina thermofila numbering) are indicated with a dot. The consensus GPxL motif representing the sequence recognition code for Homo sapiens hCypA (Piotukh K, Gu W, Kofler M, Labudde D, Helms V, Freund C (2005) Cyclophilin A binds to linear peptide motifs containing a consensus that is present in many human proteins. J Biol Chem 280:23668–23674) is underlined. (TIFF 20027 kb)

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Dimou, M., Venieraki, A., Zografou, C. et al. The cytoplasmic cyclophilin from Azotobacter vinelandii interacts with phosphate acetyltransferase isoforms enhancing their in vitro activity. Mol Biol Rep 39, 4135–4143 (2012). https://doi.org/10.1007/s11033-011-1196-1

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