Download MendeleyInsights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement.
Buller, A.R., Freeman, M.F., Wright, N.T., Schildbach, J.F., Townsend, C.A.(2012) Proc Natl Acad Sci U S A 109: 2308-2313
- PubMed: 22308359
- DOI: https://doi.org/10.1073/pnas.1113633109
- Primary Citation of Related Structures:
3S3U - PubMed Abstract:
ThnT is a pantetheine hydrolase from the DmpA/OAT superfamily involved in the biosynthesis of the β-lactam antibiotic thienamycin. We performed a structural and mechanistic investigation into the cis-autoproteolytic activation of ThnT, a process that has not previously been subject to analysis within this superfamily of enzymes. Removal of the γ-methyl of the threonine nucleophile resulted in a rate deceleration that we attribute to a reduction in the population of the reactive rotamer. This phenomenon is broadly applicable and constitutes a rationale for the evolutionary selection of threonine nucleophiles in autoproteolytic systems. Conservative substitution of the nucleophile (T282C) allowed determination of a 1.6-Å proenzyme ThnT crystal structure, which revealed a level of structural flexibility not previously observed within an autoprocessing active site. We assigned the major conformer as a nonreactive state that is unable to populate a reactive rotamer. Our analysis shows the system is activated by a structural rearrangement that places the scissile amide into an oxyanion hole and forces the nucleophilic residue into a forbidden region of Ramachandran space. We propose that conformational strain may drive autoprocessing through the destabilization of nonproductive states. Comparison of our data with previous reports uncovered evidence that many inactivated structures display nonreactive conformations. For penicillin and cephalosporin acylases, this discrepancy between structure and function may be resolved by invoking the presence of a hidden conformational state, similar to that reported here for ThnT.
Organizational Affiliation:
Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
