1.2.1. Overall structure of GNATs
Although the homology in the primary sequence between GNATs is moderate
(3%–23%), they are folded into a highly conserved three-dimensional
structure. The core secondary elements of the GNAT proteins consist of
six or seven β-strands and four α-helices, connected in order
β0-β1-α1-α2-β2-β3-β4-α3-β5-α4-β6. Four conserved motifs, known as the
N-acetyltransferase domain, are found in this core arranged in order
C-D-A-B (Figure 1C) (Dyda et al., 2000; Hentchel & Escalante-Semerena,
2015; Tercero et al., 1992).
Motifs C and D play an essential role in protein stability, while motifs
A and B contain the residues involved in acyl-CoA and acceptor substrate
binding, respectively. (Dyda et al., 2000; Vetting et al., 2005). Motif
A contains a six-residue segment (Q/R)-X-X-G-X-(G/A), known as P-loop
(phosphate-binding loop), for substrate recognition and binding and
glutamic or aspartic residue to deprotonate the target lysine (Liu et
al., 2008; Lu et al., 2017; Vetting et al., 2005). Across the entire
GNAT superfamily, there is structure divergence in the loop β1β2, the
α-4 helix of motif B, and strand β6 at the C-terminal, which together
form the binding site for the acceptor substrate. Specifically, the
C-terminal region contains a loop of varying length and position, and
the residues in the motif B are not well conserved. These structural
variations allow the recognition of various substrates (Dyda et al.,
2000; Salah et al., 2016). For example, in the mycothiol synthase
(Rv0819) from Mycobacterium tuberculosis, the β-strand 1 in the
N-terminal domain is missing, the position of helix 2, and a long loop
inserted between α3′ and β5′ (Figure 1D), while aminoglycoside
6´-N-acetyltransferase from Enterococcus faecium has an
additional α-helix between the strands β1 and β2 (Vetting et al., 2003;
Wybenga-Groot et al., 1999).
The binding of the enzyme to the substrate is through interactions with
the pantetheine and pyrophosphate moieties of CoA (Clements et al.,
1999; Dutnall et al., 1998; Hentchel & Escalante-Semerena, 2015; Lin et
al., 1999; Rojas et al., 1999;
Wang et al., 2008;). The
pantetheine binding is based on hydrogen bonds with the main chain of β4
and β 5, and the pyrophosphate is coordinated mostly by the main chain
nitrogen atoms of the conserved phosphate binding loop between β4 and α3
(Majorek et al., 2017).