Fig. 2. The mechanism of autocyclisation.

a A scheme summarising the main reaction pathways of autocyclases. After the generation of a thioester intermediate (2) from a spontaneous intramolecular rearrangement, the reaction can proceed via the following paths: (i) The unimolecular reaction—intramolecular nucleophilic attack by the N-terminal glycine amino group. This pathway results in the cyclised product (3) and free SrtA. (ii) Bimolecular reaction—intermolecular nucleophilic attack. This reaction results in a free SrtA and a protein-protein-SrtA adduct (4—tandem autocyclase). This adduct can self-cyclise to form a cyclic dimer (5). (iii) Hydrolysis—the thioester is resolved by a hydroxide ion and, irreversibly, produces a linear hydrolyzed product (5). b The reaction rates of aMSP11-L₁₂ cyclisation are compared to the equivalent reaction using the traditional (bimolecular) enzymatic cyclisation method (using MSP11 and WT SrtA in both cases). Rates were determined by monitoring the build-up of cMSP11 using LC/MS. The rates demonstrate that the autocyclase reaction at lower starting concentrations approximates first order reaction kinetics, while the bimolecular reaction approximates second order reaction kinetics. Error bars indicate standard deviations of the mean from three independent replicated experiments (individual data points shown as dots). c The MS spectrum (extracted ion chromatogram) of an aMSP11-L₁₂ cyclisation reaction at a high starting autocyclase concentration (200 μM) shows the formation of cyclised monomeric and dimeric products. Identity of the two species were confirmed by the observed m/z ion masses.