Prior to this study, the utilization of ATP hydrolysis for cyclodehydration was poorly understood. Although it had been noted that the initial steps of this transformation could mimic the mechanism of protein splicing by intein domains (an ATP-independent process; ), the absolute requirement for nucleotide triphosphate hydrolysis had never definitively been factored into the reaction2,5
. Our findings make the TOMM D-proteins only the second enzyme class known that can utilize ATP to directly activate an amide carbonyl oxygen, the other being the PurM superfamily. PurM family members conduct similar chemistry on non-peptide substrates (Supplementary Fig. 14
and in cases where crystal structures have been solved, all contain a non-canonical ATP-binding domain26,28
. Despite the similarity in ATP usage between TOMM D-proteins and PurM family members, the two enzyme families share no sequence similarity and represent an example of convergent evolution towards a common mode of ATP utilization. Members of the PurM family are proposed to act via the formation of an iminophosphate intermediate26
, whereby phosphorylation occurs prior to nucleophilic attack (Supplementary Fig. 14
). While a similar mechanism can be drawn for azoline formation by the TOMM synthetase (Supplementary Fig. 15
), it would require a disfavored 5-endo-trig cyclization29
and is inconsistent with the suppression of ATP hydrolysis in the presence of BalhA1-NC. Alternatively, we hypothesize that azoline formation by the TOMM synthetase occurs through a hemi-orthoamide intermediate analogous to that implicated in protein autoproteolytic pathways30,31
. During intein splicing and other autoproteolytic events, the hemi-orthoamide is resolved by N
-protonation and (thio)ester formation ()32
. We assert that the phosphorylation of the amide oxygen (in lieu of O
-protonation) would direct the hemi-orthoamide towards azoline formation and prevent the non-productive breakdown of the intermediate (). Phosphorylation would not only accelerate the elimination reaction (based on the lower pKa
of phosphate relative to water), but would directly couple ATP hydrolysis to cyclodehydration, providing a thermodynamic drive for the reaction. The resolution of the hemi-orthoamide via the inclusion of a thermodynamically favorable step is seen in all autoproteolytic pathways32
. Further evidence supporting a mechanism where cyclization precedes phosphorylation (i.e.
the intein-like mechanism) comes from a recent report of ester formation during microcin B17 biosynthesis33
and the discovery that engineered intein domains can catalyze azoline formation34
. In light of earlier reports, and the data presented here, TOMM cyclodehydration and intein splicing proceed through a common intermediate, which we propose is the hemi-orthoamide depicted in .
TOMM azoline installation is reminiscent of intein-mediated protein splicing
While previous studies on fused C–D proteins, implicated the YcaO/DUF181 domain in the cyclodehydration reaction, the exact role of the D-domain in this complex was never elucidated. Besides the TOMM D-protein, only one other YcaO/DUF181 family member has had a biological function reported. A recent study linked the function of E. coli
YcaO to ribosomal thiomethylation, but did not reveal any mechanistic details or physiological ramifications of this process35
. Thus, our data assign the first definitive function to a member of this uncharacterized protein family. As such, the YcaO/DUF181 protein that exists in all TOMM biosynthetic clusters has the potential to provide insight into the activity of an estimated 3000 conserved/hypothetical genes that remain largely unannotated in GenBank. At the time of writing, approximately 500 of these lie in bioinformatically recognizable TOMM clusters, all of which are likely to perform the reaction described in this report1,3
. While we cannot confidently comment on the function of the remaining 2500 genes, it is possible that non-TOMM members of the YcaO/DUF181 families harbor ATP/GTP-binding sites that either facilitate or otherwise control enzymatic activity.
Our dissection of the cyclodehydratase complex and reclassification of the roles the C/D-proteins posed an interesting question as to the role of the C-protein in TOMM biosynthesis. While we have shown that BalhC acts cooperatively with BalhD to accelerate azoline formation and govern the proper utilization of ATP, this study does not allow us to conclusively deduce the role of BalhC. Nonetheless, it is conceivable that the C-protein could partake in one of three plausible functions: (1) BalhC activates BalhD through an allosteric mechanism, increasing the rate of heterocyclization, (2) BalhC catalyzes the nucleophilic attack of the proceeding side chain by providing the requisite general acid/base residues, or (3) BalhC forms key contacts with both the substrate and BalhD to facilitate the interaction of the core region of BalhA1 and the BalhD active site. Our data do not distinguish between these possibilities, which are not necessarily mutually exclusive.
In summary, the results reported herein have not only elucidated the role of ATP in the heterocyclization of TOMM substrates, but have also implicated the hydrolysis of this co-substrate in the activation of the peptide backbone during substrate processing. Furthermore, we have obtained evidence demonstrating that the D-protein is solely responsible for this ATP-dependent cyclodehydration reaction and, as such, have assigned an enzymatic function to the previously enigmatic YcaO/DUF181 protein family. These discoveries pave the way for future work focused on uncovering the complex quaternary interactions involved in regulation of cyclodehydratase activity and the characterization of the catalytic architecture for this poorly understood ATP-utilizing enzyme.