(a) The TOMM clusters from Bacillus sp. Al Hakam (Balh) and Escherichia coli (Mcb) are depicted along with the percent amino acid identity for each of the three key proteins. Gene assignments are given below. Note that the dehydrogenase and the “cyclodehydratase” in the microcin B17 cluster are assigned as McbC and McbB, respectively. The sequences of the peptide substrates used in this study are shown. Color-coding: green, point mutations; orange, residues known to be cyclized in vitro blue hyphen, putative leader sequence cleavage site; blue caret, known leader sequence cleavage site. (b) Heterocycles are installed on a precursor peptide by a heterotrimeric complex composed of a “cyclodehydratase” (C), a “docking/scaffolding” protein (D) and a dehydrogenase (B). The cumulative mass change for each step is shown below the modification. (c) The two leading hypotheses for ATP utilization. ATP hydrolysis could be used to control conformational dynamics (molecular machine) or to directly activate the peptidic substrate as shown. Reactions carried out in [¹⁸O]-H₂O will give different products, allowing for distinguishing between these mechanisms. X = S, O; R = H, CH₃.

(a) MALDI-TOF spectra monitoring heterocycle formation. Labels represent the component omitted from the reaction (e.g. –C lacked BalhC). The mass shift for the major species relative to the unmodified substrate is displayed above the appropriate peak. The stoichiometry of ATP hydrolysis to azole heterocycle formation in (b) the Balh TOMM and (c) the Mcb TOMM is shown. Phosphate (squares), heterocycles (circles), BCD background ATPase activity (solid line, PNP assay; dashed line, extrapolated PNP assay; triangles, malachite green assay), maximum product produced based on the concentration of substrate (solid horizontal line). Error bars represent the standard deviation from the mean (n=3). The ATP/azole stoichiometric ratio is displayed and is the average of all time points.

³¹P-NMR spectra of reactions with (a) the Balh or (b) the Mcb synthetase. The identity of the major product, [¹⁶O]-P_i, was confirmed by spiking with authentic [¹⁶O]-P_i. Slight pH differences between the Balh and Mcb samples (8.5 and 8.0, respectively) account for the altered chemical shifts. (c) MALDI-TO-MS spectra of BalhA1 treated with a stoichiometric amount of either BalhC or BalhD are displayed. The +1 charge state is shown. Mass shifts relative to the BalhA1 control are labeled. (d) A ³¹P-NMR spectrum of a reaction conducted in [¹⁸O]-water confirmed the presence of both [¹⁶O₄]-P_i and [¹⁸O₁,¹⁶O₃]-P_i (bottom). The identity of the [¹⁶O₄]-P_i peak was confirmed by spiking the sample with authentic [¹⁶O₄]-P_i (top).

(a) The mechanism of protein splicing and autoproteolysis follows the N-protonation pathway to generate a (thio)ester. An azoline heterocycle could also be formed from the hemi-orthoamide intermediate if O-protonation occurred. (b) In TOMM biosynthesis, the use of ATP to phosphorylate the substrate could be used to drive the reaction down an O-elimination pathway to generate an azoline. X = S, O; R = H, CH₃.