4IHD

Crystal Structure of Uncleaved ThnT T282C, derivatized at the active site with EtHg

Summary for 4IHD

• Entry DOI: 10.2210/pdb4ihd/pdb
• Related: 3S3U 3TM1 3TM2 4IHE
• Descriptor: ThnT protein, ETHYL MERCURY ION (3 entities in total)
• Functional Keywords: dom-fold, clan pe, family p1, autoproteolysis, pantetheine hydrolase, thienamycin biosynthesis, ethylmercury derivatization of c282, hydrolase
• Biological source: Streptomyces cattleya
• Total number of polymer chains: 2
• Total formula weight: 83207.54

Authors

Buller, A.R.,Schildbach, J.F.,Townsend, C.A. (deposition date: 2012-12-18, release date: 2014-08-13, Last modification date: 2023-09-20)

Primary citation

Buller, A.R.,Freeman, M.F.,Schildbach, J.F.,Townsend, C.A.
Exploring the Role of Conformational Heterogeneity in cis-Autoproteolytic Activation of ThnT.
Biochemistry, 53:4273-4281, 2014

PubMed 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.

PubMed: 24933323
DOI: 10.1021/bi500385d

Experimental method

X-RAY DIFFRACTION (1.65 Å)