Molecular dynamics explorations of active site structure in designed and evolved enzymes

Resumen

Conspectus This Account describes the use of molecular dynamics (MD) simulations to reveal ho mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient hen functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. e describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations beteen active and inactive conformations normally concealed to static crystallography. MD shos ho the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. e first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. e find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, e sho ho microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme as evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution shoed that the active site of the ild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP, a noncatalytic arrangement of the catalytic triad is dominant. Unnatural truncated substrates are inactive because of the lack of protein-protein interactions provided by the ACP. Directed evolution is able to gradually restore the catalytic organization of the active site by motion of the protein backbone that alters the active site geometry. In the third case, e demonstrate the key role of MD in combination ith crystallography to identify the origins of substrate-dependent stereoselectivities in a number of Codexis-engineered ketoreductases, one of hich is used commercially for the production of the antibiotic sulopenem. Here, mutations alter the shape of the active site as ell as the accessibility of ater to different regions of it. Each of these examples reveals something different about ho mutations can influence enzyme activity and shos that directed evolution, like natural evolution, can increase catalytic activity in a variety of remarkable and often subtle ays. © 2015 American Chemical Society.