FIGURE 2.

Enzymatic protein ac(et)ylation is catalyzed by GNAT ac(et)yltransferases in bacteria. (A) Structure of the E. coli GNAT acetyltransferase RimI in complex with acetyl-CoA (PDB: 2CNS). All bacterial protein acetyltransferases belong to the GNATs. These are characterized by sequence motifs A-D as indicated. Motifs A and B are important for CoA-binding. Motif A contains the characteristic sequence motif Arg/Gln-x-x-Gly-x-Gly/Ala (x: any amino acid), known as P-loop, which contacts the phosphates of the acetyl-CoA/CoA. The acetyl-CoA is shown in ball-and-stick representation [the figure was generated with PyMOL v.2.3.4 (Schrödinger LLC, 2000)]. (B) Domain organization of different bacterial GNAT types. Type I GNATs contain an N-terminal and type II GNATs a C-terminal NDP-forming acyl-CoA synthetase domain. These domains are catalytically inactive, but they bind acetyl-CoA and are important for allosteric regulation of GNAT activity. Type III GNATs encompass an N-terminal ligand binding domain such as a cAMP-binding domain with high similarity for cAMP-binding domains of EPAC, PKA, CAP/CRP, a Rossmann-fold domain specific for NADP⁺-binding, or an ACT domain for binding to amino acids cysteine, arginine and/or asparagine. Binding of these metabolic molecules to the N-terminal domain activates the C-terminal GNAT activity. Type IV GNATs contain only the catalytic GNAT domain and no accessory domain. Type V GNATs are composed of a tandem GNAT and a C-terminal region important for oligomerization and catalytic activity. Only the N-terminal GNAT domain is active, the central GNAT domain is important for structural integrity. (C) Several mechanisms contribute to catalytic activity of bacterial GNATs. Mammalian GNATs are shown to use a general base catalytic mechanism for acetyl-group transfer. In bacteria, not all GNATs contain a catalytic glutamate acting as general base and other mechanisms contribute to catalysis. The electrostatics in the active site might favor substrate amino group deprotonation. Some GNATs use a glutamate as general base for deprotonation of the substrate amino group. Other GNATs were reported to use a remote base, such as an activated serine residue to orient and polarize a catalytic water molecule acting as general base during catalysis. Other GNATs were reported to use an serine residue as catalytic base after activation by an active site water molecule. This catalytic strategy involves the formation of a serine-bound acetyl-enzyme intermediate. (D) Catalytic mechanism exerted by GNATs using a general base catalyst. Many GNATs use a catalytic glutamate as general base that abstracts a proton from the substrate amino group increasing its nucleophilicity for attack of the electrophilic carbonyl carbon of ac(et)yl-CoA. A tetrahedral intermediate is formed, which is resolved to yield the ac(et)ylated substrate amino group and CoA. For some GNATs an active site tyrosine contributes as catalytic acid resolving the tetrahedral intermediate by protonating the sulfhydryl group of the leaving CoA [figure redrawn and modified from Ali et al. (2018) and Blasl et al. (2021)].