Abstract
Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.
Highlights
PatZ is the main Escherichia coli acetyltransferase and controls acetyl-CoA synthetase (Acs) activity
These data showed that the enzymatic rate was slightly higher for the substrate Acs than for acetyl-CoA, whereas the K0.5 was much higher for acetyl-CoA
Positive cooperativity is unusual in enzymes belonging to the GCN5related N-acetyltransferase (GNAT) family, this behavior has been described in the PatZ homologous enzyme, SePat, with a Hill coefficient of 2.2 Ϯ 0.2 [8]n suggesting that the structural basis for this behavior is protein tetramerization in the presence of acetyl-CoA
Summary
PatZ is the main Escherichia coli acetyltransferase and controls acetyl-CoA synthetase (Acs) activity. We demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. This modification is reversed by the sirtuin CobB. An in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism
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