cis-Autoproteolysis is a post-translational modification necessary for the function of ThnT, an enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. This modification generates an N-terminal threonine nucleophile that is used to hydrolyze the pantetheinyl moiety of its natural substrate. We determined the crystal structure of autoactivated ThnT to 1.8 Å through X-ray crystallography. Comparison to a mutationally inactivated precursor structure revealed several large conformational rearrangements near the active site. To probe the relevance of these transitions, we designed a pantetheine-like chloromethyl ketone inactivator and co-crystallized it with ThnT. Although this class of inhibitor has been in use for several decades, the mode of inactivation had not been determined for an enzyme that uses an N-terminal nucleophile. The co-crystal structure revealed the chloromethyl ketone bound to the N-terminal nucleophile of ThnT through an ether linkage, and analysis suggests inactivation through a direct displacement mechanism. More importantly, this inactivated complex shows that three regions of ThnT that are critical to the formation of the substrate binding pocket undergo rearrangement upon autoproteolysis. Comparison of ThnT with other autoproteolytic enzymes of disparate evolutionary lineage revealed a high degree of similarity within the proenzyme active site, reflecting shared chemical constraints. However, after autoproteolysis, many enzymes, like ThnT, are observed to rearrange in order to accommodate their specific substrate. We propose that this is a general phenomenon, whereby autoprocessing systems with shared chemistry may possess similar structural features that dissipate upon rearrangement into a mature state.
Bibliographical noteFunding Information:
We gratefully acknowledge Vivian Stojanoff and Jean Jackoncic at the National Synchrotron Light Source for assistance in data collection. N-terminal sequencing was performed by Jodie Franklin at the Johns Hopkins Medical School Sequencing Facility. This work was supported by research grants National Institutes of Health RO1 AI014937 (to C.A.T.) and National Institutes of Health GM61017 (to J.F.S.) and the Dimitri V. d'Arbeloff post-doctoral fellowship (to N.T.W.).
- N-terminal nucleophile
- X-ray crystallography
- mechanism of chloromethyl ketone inhibition
- thienamycin biosynthesis