Figure 1.
Acyltransferase reaction mechanism and sequence logos.
A) AT reaction mechanism: ATs utilize a ping-pong mechanism to transfer acyl groups from CoA to ACP (for most ATs, R1 = H or CH3). Hydrolysis, shown in the bottom branch is a competing, unproductive reaction. B) Sequence logo for the motif containing the active site serine of pfam PF00698, which encompasses ATs from fatty acid and polyketide biosynthesis. C) Sequence logo for pfam PF01734, which are evolutionarily related phospholipases. Logos created using Skyline [12].
Figure 2.
Acylation and transacylation activities of WT, H640A, S641A, and H640A+S641A.
A) Transacylation activity as observed by high-resolution LC/QTOFMS. Data for the variants (WT, H640A, S641A, H640A+S641A) incubated with malonyl-CoA and a wildtype negative control without malonyl-CoA are shown (WT – Mal-CoA). B) Formation of malonyl-AT complex for wildtype (monoisotopic peptide m/z = 1000.9903, z = 4) and H640A (monoisotopic peptide m/z = 984.4849, z = 4) as observed by high-resolution LC/QTOFMS. Data for wildtype and H640A (WT, H640A) incubated with malonyl-CoA as well as a wildtype negative control without malonyl-CoA (WT – Mal-CoA) are shown. The mass for each chromatogram is shown in parenthesis to the right. Additional details on chromatogram preparation in the Methods S1.
Table 1.
Hydrolysis rates for yersiniabactin AT mutants at a concentration of 35 µM malonyl-CoA.
Figure 3.
Crystal structure with malonate of the acyltransfserase from DynE8, an iterative type I PKS.
Residues involved in catalysis are labeled and shown as sticks. The hydrogen bonding water highlighted in the text is shown as a sphere. Figure 3 was prepared using Pymol from the PDB entry 4AMP [11].