Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation

Abstract

The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.

Figure 1

Topology scheme of the GNAT proteins. Starting at the N-terminal end, the secondary structure elements are colored dark green (β0, β1, α1, and α2), yellow (β2–β4), red (α3 and β5), and cyan (α4 and β6).

Figure 2

General structures of various aminoglycosides. The amino-cyclitol (2-deoxystreptamine) is colored blue. Arrows indicate the positions that are commonly modified by AACs. (A) 4,6-Disubstituted deoxystreptamine aminoglycoside. Examples cited in this review are butirosin, paromomycin, and neomycin. (B) 4,5-Disubstituted deoxystreptamine aminoglycoside. Position 2′ can be either an amino group (kanamycin B, gentamicin C1a and C1, etc.) or a hydroxyl group (kanamycin A, amikacin, etc.).

Figure 3

Structures of aminoglycoside N-acetyltransferases. The secondary structure elements are colored like the topology scheme presented in Figure 1. The substrates or products bound are rendered as sticks with white carbons. (A) Structure of S. marcescens AAC(3)-Ia (PDB entry 1BO4). (B) Structure of M. tuberculosis AAC(2′)-Ic in complex with CoA and kanamycin A (KAN) (PDB entry 1M4I). (C) Superimposition of the S. enterica AAC(6′)-Ib structure in complex with CoA and its variant AAC(6′)-Ib₁₁ (PDB entries 2PRB and 2PR8, respectively). The α-helical flap (highlighted with an arrow in each structure) located above the aminoglycoside binding site is colored purple in the AAC(6′)-Ib structure and orange in the AAC(6′)-Ib₁₁ structure.

Figure 4

Role and structure of MshD in mycothiol biosynthesis. (A) Biosynthetic pathway of mycothiol. The box highlights the reaction catalyzed by MshD. (B) Structure of M. tuberculosis MshD in complex with CoA and DAM (PDB entry 2C27). The secondary structure elements are colored like the topology scheme presented in Figure 1. The substrates or products bound are rendered as sticks with white carbons. (C) Superimposition of MshD in complex with CoA and DAM (PDB entry 2C27, gray) and MshD in complex with AcCoA (PDB entry 1OZP, beige). Upon substrate binding, the N-terminal domain rotates closer to the C-terminal domain to allow a narrower binding cavity for DAM as highlighted by the arrow.

Figure 5

Scheme of peptidoglycan layer formation. Fem enzymes act at different stages of the peptide bridge formation depending on the bacterial species. In W. viridescens, FemX adds only the first amino acid. In S. aureus, FemX adds the first glycine residue whereas FemA adds the second and third glycine residues and FemB the last two glycine residues. For the sake of simplicity, only three of five amino acids in the peptide bridge are represented in the figure. After peptide bridge formation, the terminal Gly₅ α-NH₂ group of the peptide bridge is cross-linked to the penultimate amino acid of the neighboring pentapeptide stem by transpeptidation leading to the release of the terminal amino acid from the stem peptide.

Figure 6

Structural comparison of W. viridescens FemX and S. aureus FemA. The GNAT secondary structure elements are colored like the topology scheme presented in Figure 1. The bisubstrate is rendered as sticks with white carbons. (A) The W. viridescens FemX consists of two similar GNAT domains, 1A and 1B (PDB entry 1P4N). (B) The S. aureus FemX structure also displays domains 1A and 1B and contains an additional coiled coil segment that is colored orange (PDB entry 1LRZ).

Figure 7

Structures of Rim proteins. The secondary structure elements are colored like the topology scheme presented in Figure 1. CoA and the bisubstrate are rendered as sticks with white carbons. (A) Structure of S. tphimurium LT2 RimL in complex with CoA (PDB entry 1S7N). (B) Structure of S. typhimurium LT2 RimI in complex with a bisubstrate CoA-S-acetyl-S18_1–6 (PDB entry 2CNM).

Figure 8

Activation of M. tuberculosis Pat via cAMP binding (left structure, PDB entry 4AVA; right structure, PDB entry 4AVC). The N-terminal regulatory domain (orange) is linked to C-terminal catalytic GNAT domain. Upon cAMP binding, the Pat protein undergoes a conformational rearrangement allowing protein–substrate binding in the catalytic domain active site. The GNAT secondary structure elements are colored like the topology scheme presented in Figure 1. The substrates and activators bound are rendered as sticks with white carbons.