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Microbial Pathogenesis: A GNAT from Pseudomonas

SBKB [doi:10.1038/sbkb.2012.183]
Featured Article - February 2014
Short description: A multidisciplinary approach offers a detailed view of a bacterial acetyltransferase.

Structure of PA4794 in complex with reaction products CoA and Nε-acetylated NPAcGK peptide. (PDB: 4L8A) Interaction of the C-terminal carboxyl group of the peptide with Arg49 of PA4794 is visible. Figure courtesy of Wladek Minor.

Gnc5-related N-acetyltransferases (GNATs) are a superfamily of enzymes that transfer an acetyl group from acetyl-coenzyme A (CoA) to a wide range of substrates, including proteins and small molecules such as antibiotics and spermine. In proteins, GNATs have been found to modify the N-terminus (Nα) or internal lysine residues (Nε).

In contrast to eukaryotic GNATs, the functions of bacterial GNATs have not been well studied. Minor and colleagues (PSI MCSG and CSGID) now present a comprehensive characterization of PA4794, an enzyme from Gram-negative bacterium Pseudomonas aeruginosa, using a combination of biochemical, biophysical and crystallographic approaches.

PA4794 was initially crystallized in its apo form, as well as in complex with acetyl-CoA and CoA, which confirmed its similarities to other GNATs and revealed conformational changes that take place upon CoA binding. The authors next sought to identify the substrates for PA4794's acetyltransferase activity. A biochemical screen showed that the enzyme could acetylate L-lysine and derivatives such as Nα-Ac-L-lysine, but not Nε-acetyl lysine.

The data revealed a strong preference for the peptide N-phenylacetyl-Gly-Lys (NPAcGK), so the authors soaked crystals of apo-PA4794 with acetyl-CoA and the peptide. The structure showed the products of the reaction, namely CoA and the peptide acetylated at lysine's Nε. Here, the authors observed that the C-terminal carboxyl group of the peptide was stabilized by multiple interactions. By testing the enzyme with different peptides, they found that a free carboxyl group on Lys is important for enzymatic activity.

Finally, the catalytic mechanism of PA4794 was investigated in detail by mutagenesis and kinetic studies. The authors also identified several small molecules with inhibitory activity on PA4794, including cephalosporins. Enzymatic, isothermal calorimetry and crystallographic analyses revealed that the cephalosporin compounds act as competitive inhibitors and mimic the conformation of the acetylated peptide product.

These findings indicate that PA4794 is an acetyl transferase specific for peptides with C-terminal lysine, an activity that had not been described previously, though the identity of biological substrates of PA4797 remains to be determined. P. aeruginosa is an opportunistic pathogen that causes nosocomial infections notoriously difficult to treat, due to the bacterium's low susceptibility to antibiotics. Thus, the finding that PA4794 is inhibited by cephalosporins may have important clinical implications.

Inês Chen

References

  1. K.A. Majorek et al. Structural, functional and inhibition studies of a GNAT superfamily protein PA4794: a new C-terminal lysine protein acetyltransferase from Pseudomonas aeruginosa.
    J Biol Chem. 288, 30223-35 (2013). doi:10.1074/jbc.M113.501353

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