Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates

Abstract

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates.

Role of conformational dynamics in the evolution of novel enzyme function

“…Using tICA as a dimensional reduction technique, we constructed the associated free energy landscape revealing that the wild-type enzyme can display four major conformational states. 85 The analysis of these conformational states in combination with active site volume calculations, provided evidence that the most populated wild-type conformations, in which the catalytic machinery is well-positioned for catalysis, 86 present small active site pocket volumes (see Fig. 5 ).…”

Stability of ligand-induced protein conformation influences affinity in maltose-binding protein

“…We are no longer limited to static high-resolution structures but can also probe the conformational dynamics of a protein, for instance by single-molecule FRET. The simplest but somewhat naive way to explain ligand affinity deals with the architecture of the binding pocket, which needs to allow for enough protein-ligand interactions and proper orientation of the ligand (4,13,43,44). Differences in ligand affinity of homologous proteins have been explained by differences in binding pocket organization (38,45).…”

“…Advances in the fields of electron paramagnetic resonance (EPR) (7-9), molecular dynamics (MD) (5,(8)(9)(10)(11)(12)(13) and single-molecule spectroscopy (14)(15)(16)(17)(18)(19) allow visualization of protein conformational dynamics on a femtosecond to even second time scales. This led to increased appreciation of the concept that different protein conformations are a determinant of ligand affinity and catalytic activity.…”

Enzyme Dynamics: Looking Beyond a Single Structure

“…It is however possible following a dimensionality reduction by tICA to elegantly present the various conformations as energy landscapes . This method was recently used to explain the ability of epoxide hydrolase from Bacillus megaterium ( Bm EH) to hydrolyze various epoxide substrates, including its acceptance of bulky substrates . Four accessible conformations were identified, three of which shared a bigger active‐site cavity, which allows the bulkier substrates access to the active‐site, with one remaining catalytically unproductive.…”

“…However, this latter conformation is separated from the other three by a 3 kcal/mol energy barrier. The authors also uncovered that two mutants which can process bulkier substrates use partial unfolding of both the lid domain and the α helix bearing the catalytic Tyr144 in order to increase the volume of the catalytic pocket even further than the wild type (WT) enzyme …”