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Title: Structural study of the adenylation domain by molecular dynamics simulation
Author: Thalassinou, Joanne Frances
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2012
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As antibiotic resistance is increasing more rapidly than new antibiotics are produced and/or discovered, there is an increasing need to identify new ways to design novel antibiotics. A potential avenue for this, is the exploitation of Nonribosomal Peptide Synthetases (NRPSs) from bacteria and fungi which biosynthesise structurally complex biologically active peptide products, including numerous potential antibiotics and other molecules with pharmacologically attractive properties. In order to do so, however, a detailed molecular understanding of NRPSs is required. NRPSs are modular proteins, with each module comprising domains that each perform specific functions to select, activate, alter (optional) and combine amino/hydroxyl acid substrates to form a specific peptide product. The Adenylation domain (A domain) specifically selects and activates the substrate through a two step reaction. In the first half reaction, a highly reactive aminoacyl adenylate is formed by reaction with Mg-adenosine triphosphate (ATP) resulting in the release of pyrophosphate. In the second half reaction the A domain binds the phosphopantetheinyl (PPant) arm of the downstream domain, the Peptidyl Carrier Protein (PCP) domain. The terminal thiol of the PPant arm attacks the activated aminoacyl group displacing adenosine monophosphate (AMP), leaving the amino acid substrate tethered to the PCP domain as a thioester. The A domain is of particular interest as a target for engineering approaches as it is considered to be the primary determinant of substrate specificity. Little is understood, however, about the molecular basis of substrate selectivity or how the dynamics of the domain enable the two part reactions to take place. In 1997, the first A domain structure was determined; the L-phenylalanine (L-Phe) activating A domain (PheA) of the Gramicidin S synthetase from Bacillus brevis. All of the A domain structures determined to date are either unligated (apo form) or co-crystallised with reactants or products from the first half reaction. The NRPS A domains are members of the adenylate-forming superfamily which have been structurally characterised in three states, apo, with the first half reaction and second half reaction ligands. Comparison between these structures, suggested these enzymes use a domain alternation strategy to reconfigure a single active site to perform two different reactions. While the A domains have only been determined in the adenylate-forming conformation, similarities between members of the adenylate-forming superfamily suggest NRPS A domains may exploit of a similar strategy of domain alternation to reconfigure the enzyme’s single active site. To date, no molecular simulation study of any NRPS A domain has been reported in the literature. In this study, molecular dynamics (MD) simulations of the PheA have been carried out in the apo form, with the cognate substrate, and with noncognate substrates, to understand the molecular basis of substrate specificity and the effect of the substrate on the dynamics of the protein. Inter-domain rotation was observed in the apo and cognate holo simulations and with one of the noncognate substrates, L-Thr. This motion occurred between the Acore domain and Asub domain or part of the Asub domain. The rotation observed in the simulations with the cognate substrate creates a widening between the two domains of PheA on the side of the enzyme where the PPant arm is thought to bind. Results from one of the cognate holo simulations suggests the A3 motif loop may be important in stabilising the A domain to increase the domain rotation or maintaining the opening through with PPant is proposed to access the active site. Results from one of the noncognate substrate simulations, L-Asp substrate, suggests a role for the A3 motif loop in the removal of noncognate ligands from the binding site. Results from the simulation with noncognate substrate L-Tyr also suggest that interaction of the substrate with the key Asp and Lys binding pocket residues may be required for rotation of the Asub domain can occur. A homology model of the second A domain of the NRPS that forms Coelichelin has built and it is shown that the core regions of the model are stable in the MD simulations carried out in the apo form, with the cognate ligand (L-Thr) and noncognate ligands (L-Ser and L-Val). Some domain rotation was observed in the simulations with L-Thr and L-Ser. The findings from this study support the suggestion that interaction between the key Asp and Lys binding pocket residues and the substrate may be required for domain rotation. This work presented in this thesis useful insight into the dynamics of the A domain and provides evidence for the role of the conserved A3 motif loop in both domain rotation and removal of noncognate ligands from the binding pocket.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry