Structure-function analyses on AcuB encoded by the Bacillus subtilis acuABC-operon involved in regulation of acetyl-CoA synthetase by post-translational lysine acetylation

• Applicant: Professor Dr. Michael Lammers
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 534243417

Bacterial cells have to cope with rapidly changing environmental conditions, including alterations in nutrient availability. A way to sense the cellular metabolic state is the regulation of protein function by post-translational lysine acetylation. In bacteria, lysine acetylation is enzymatically catalyzed by lysine acetyltransferases (KATs) that use acetyl-CoA as donor molecule for the acetylation of lysine side chains. Acetylated lysine side chains can be deacetylated by deacetylases, which are classified into two groups, i.e. the classical Zn2+-dependent deacetylases (KDACs) and the NAD+-dependent sirtuins. Next to its role as donor molecule for acetylation, acetyl-CoA is a central metabolite. Acetyl-CoA can be, amongst other, generated by the activity of AMP-forming acetyl-CoA synthetases (Acs). This reaction is split into two half reactions. In the 1st half reaction, the adenylation reaction, acetate is activated by adenylation to yield acetyl-AMP. Subsequently, in the 2nd half reaction, acetyl-AMP is converted to the thioester acetyl-CoA (thioester formation). In the Gram-positive bacterial species Bacillus subtilis, the acetyl-CoA synthetase AcsA is encoded upstream from the acuABC-operon and is reversely transcribed. The acuABC-operon was originally identified to be involved in butanediol and acetoin catabolism. The function of the gene products of the acuABC operon are mechanistically only partially understood. The gene acuA encodes for a GNAT-type KAT to acetylate AcsA at a C-terminal lysine side chain, i.e. K549, and thereby inactivate AcsA activity. AcuC constitutes a classical Zn2+-dependent deacetylase. It is known that AcuC is capable to deacetylate and thereby (re-)activate AcsA. The exact molecular mechanisms underlying the inactivation of AcsA activity by AcuA mediated K549-acetylation are only marginally understood. The role of AcuB in the interplay of AcuA, AcuC and AcsA is totally unclear. Interestingly, other bacterial species such as Staphylococcus aureus share a similar gene organization. However, in S. aureus the operon encodes only for AcuA and AcuC but the acuB gene is missing. This suggests that AcuB plays a role that might be specific for some bacterial strains. We postulate that AcuB, which is completely functionally uncharacterized, might be an important regulator for this interplay, i.e. sensing the metabolic state, adjusting AcuA and/or AcuC activity to modulate AcsA activity. In B. subtilis, AcuB by binding to AcuC and/or AcuA and to molecules such as NAD+, AMP, ATP or CoA/acetyl-CoA might be the regulator that adapts the activity of the deactylase AcuC and/or the acetyltransferase AcuA to the cellular metabolic state enabling to coordinate AcsA activity. In this project we want to focus on the exact functional role of AcuB that is encoded by the acuABC-operon in B. subtilis. These data will reveal if regulation of classical deacetylases by binding to sensory proteins is a more general regulatory system.

• DFG Programme: Research Grants