Acyl group transfer

This model is integrated with the existing x-ray structure of the SGNH domain and published mutational data to allow additional structure-function analysis. [See – The simple two-step pattern for 7 key reactions of aldehydes and ketones]

The carbon of a C=O group is electrophilic and reacts readily with the negatively charged nucleophile through an addition mechanism [Step 1, form C-Nu, break C-O (pi) ]

Now what?

Using the CHARMM-GUI membrane builder module (Lee et al., 2019), this structure was embedded within a model E. Some of these reactions require AT3-only proteins, others are carried out by proteins with an AT3 domain fused with a C-terminal SGNH domain. aureus (an AT3-SGNH protein) are able to hydrolyse the acetyl group from acetyl-CoA and transfer it to the peptidoglycan-like acceptor substrate, effectively observing the entire catalytic cycle in vitro (Jones et al., 2021).

(See: Acid-Base Reactions Are Fast)

So the first thing that happens is not addition, but deprotonation of the carboxylic acid to give a carboxylate.

If nucleophilic acyl substitution were to happen at this point, it would have to add to this carboxylate which would make a di-anion with two negative charges on the same molecule (if that sounds unstable –  it is!!).

Furthermore, the leaving group to make the ester would not be HO(-).

It would be O (^2-) !

With one notable exception [Note 6] , that won’t happen.

Note that carboxylic acids can be converted to esters, but that the reaction requires acid (e.g.

Each acceptor substrate may present its own unique challenges for the AT3 domain. The structure of OafB consists of two key domains: the AT3 domain and SGNH domain (Figure 1C). The RaptorX model of the transmembrane domain (TMH1–11 of OafB, residues 1–376) was embedded in a model E.

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enterica subsp. One such protein, OatA, is an AT3 protein with attached SGNH domain (AT3-SGNH), that acetylates the MurNAc residue in peptidoglycan (Bera et al., 2005; Aubry et al., 2011). With enough heat, it is possible to perform the basic hydrolysis of amides, which has the appearance of being “uphill” since NH₂(-) is a stronger base than HO(-).

Overall, the study is valuable as it will help stimulate specific experimental analyses that will further evaluate and improve the model for better mechanistic understanding of this class of enzymes.

The fatty membrane that surrounds cells is an essential feature of all living things.

Using VMD as a visual tool to guide positioning, the acetyl group of acetyl-coA was placed beneath the gap between TMHs 9 and 10, and the N-terminus of the OafB₁₀₀ protein. Pulling at a rate of 0.5 nm ns^–1 and with a harmonic force of 1000 kJ mol^–1 nm^–2, the sulphur of the acetyl-CoA molecule was pulled upwards, towards the centre of mass of the alpha carbons of residues E243, D126, and E189 (all in loops at the periplasmic surface of the transmembrane domain, surrounding the pore) until the z-separation of the two groups was 0 (7.5 ns).

Three variations on the final frame of the SMD were generated in VMD by rotating and translating acetyl-coA within this pore to use as starting conformations for equilibrium MD.

These systems were energy minimised using the steepest descent algorithm to resolve steric clashes, and equilibrated as described above, with additional position restraints (Table 5) on the acetyl-coA molecule. In addition to the Arg14 coordinating the phosphate of 3’-phosphate acetyl-CoA, we noted that the conserved Arg74 (of the RXXR motif of TMH3) along with Lys279 and Arg338 formed a small pocket capable of orienting such that they can all simultaneously hydrogen bond to the 3’-phosphate group (Figure 5B).

Furthermore, energy decomposition analysis (EDA) using ONETEP (Skylaris et al., 2005; Prentice et al., 2020) indicates strong, attractive interactions between the AT3 domain and acetyl-CoA.

Given the conformational mobility of the 4’-phosphopantetheine segment of the coenzyme, and of the loop between TMH 5 and 6, it is not unreasonable to suggest that the thioester bond is accessible to both His25 and Tyr194 to aid the acetyl transfer in a mechanism such as that suggested by Jones et al., 2021.

Furthermore, standalone AT3 proteins contribute an acyl group in the biosynthesis of macrolide antibiotics in Streptomyces species which increases antibiotic efficacy (Hara and Hutchinson, 1992; Arisawa et al., 1995; Arisawa et al., 1994; Epp et al., 1989). However, the mechanism is currently largely unknown. OafB specifically O-acetylates the rhamnose moiety of this O-antigen repeating unit.

AT3 domains are predicted to have 10 transmembrane helices (TMHs; Allison and Verma, 2000; Bernard et al., 2011; Bohin, 2000; Bonnet et al., 2017; Bontemps-Gallo et al., 2016; Cogez et al., 2002; Corvera et al., 1999; Geno et al., 2017; Kajimura et al., 2006; Kintz et al., 2015; Lacroix et al., 1999; Menéndez et al., 2004; Moynihan and Clarke, 2011; Slauch et al., 1996; Spencer et al., 2017; Thanweer et al., 2008; Zou et al., 1999; Buendia et al., 1991; Bera et al., 2005); however, despite the wide-ranging functions of this family of proteins, there are currently no models for their overall structure and only limited information on mechanism.

The overall understanding of AT3 proteins is mainly derived from studies of these proteins in the context of bacterial virulence through changes on the cell surface (Vora et al., 2013; Dzitoyeva et al., 2003).

Residues 377–379 were added to the N-terminus of the SGNH domain using MODELLER 10.0 (Webb and Sali, 2016), and a peptide bond was generated between residues 376 and 377 in ChimeraX (Pettersen et al., 2021) with varying C-N-C_α-C dihedral angles (80, 100, 120, 140°) to generate proteins with differing relative conformations of the AT3 and SGNH domains (henceforth referred to as OafB₈₀, OafB₁₀₀, OafB₁₂₀, and OafB₁₄₀).