title: Exploring the role of conformational heterogeneity in cis-autoproteolytic activation of ThnT.
abstract: In the past decade, there have been major achievements in understanding the relationship between enzyme catalysis and protein structural plasticity. In autoprocessing systems, however, there is a sparsity of direct evidence of the role of conformational dynamics, which are complicated by their intrinsic chemical reactivity. ThnT is an autoproteolytically activated enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. Conservative mutation of ThnT results in multiple conformational states that can be observed via X-ray crystallography, establishing ThnT as a representative and revealing system for studing how conformational dynamics control autoactivation at a molecular level. Removal of the nucleophile by mutation to Ala disrupts the population of a reactive state and causes widespread structural changes from a conformation that promotes autoproteolysis to one associated with substrate catalysis. Finer probing of the active site polysterism was achieved by EtHg derivatization of the nucleophile, which indicates the active site and a neighboring loop have coupled dynamics. Disruption of these interactions by mutagenesis precludes the ability to observe a reactive state through X-ray crystallography, and application of this insight to other autoproteolytically activated enzymes offers an explanation for the widespread crystallization of inactive states. We suggest that the N→O(S) acyl shift in cis-autoproteolysis might occur through a si-face attack, thereby unifying the fundamental chemistry of these enzymes through a common mechanism.
authors.affiliation: Department of Biophysics, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Buller
authors.forename: Michael F
authors.initials: MF
authors.lastname: Freeman
authors.forename: Joel F
authors.initials: JF
authors.lastname: Schildbach
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D009154:Mutation; D059748:Proteolysis; D013845:Thienamycins / Q000096:biosynthesis
chemical_list: D013845:Thienamycins; D000581:Amidohydrolases; C114020:beta-lactam synthetase