The role of distant mutations and allosteric regulation on LovD active site dynamics

Abstract

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

“…The native substrate of the acyl-transferase LovD is covalently bound to an acyl carrier protein, LovF, which also functions as an allosteric activator. LovD was subjected to nine rounds of directed evolution for altered substrate specificity, thermal stability, and tolerance to organic solvent to generate a biocatalyst for production of the drug simvastatin (37). Insight into whether increases in activity were related to allosteric regulation was gleaned from microsecond molecular dynamics simulations, which suggested that LovF promotes the stability of a closed and catalytically active conformation of LovD and that directed evolution identified mutations that increased activity in a similar fashion (37).…”

“…LovD was subjected to nine rounds of directed evolution for altered substrate specificity, thermal stability, and tolerance to organic solvent to generate a biocatalyst for production of the drug simvastatin (37). Insight into whether increases in activity were related to allosteric regulation was gleaned from microsecond molecular dynamics simulations, which suggested that LovF promotes the stability of a closed and catalytically active conformation of LovD and that directed evolution identified mutations that increased activity in a similar fashion (37). Our kinetic, structural, and spectroscopic data provide firm experimental support for a similar effect in TrpB, demonstrating that allosteric potential can be readily converted into catalytic activity through directed evolution.…”

Exploring the potential impact of an expanded genetic code on protein function

“…In addition, structural and dynamic studies of dihydrofolate reductase have shown that mutations that affect millisecond-time-scale fluctuations lead to decreased rates of hydride transfer (36). Similarly, crystallographic analysis and microsecond-scale molecular-dynamics simulations have suggested that remote mutations in simvastatin synthase alter catalytic efficiency through conformational effects (37). Other examples include the demonstration of altered catalytic activity in conformational isomers of a catalytic antibody and the effects of distal mutations on HIV protease active-site conformation and catalytic activity (38).…”

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

“…Moreover, MD can help in the future to discern long range effects. In fact, it has been recently used to highlight the impact of distant mutations on active site preorganization in evolved enzymes [159,160]. Furthermore, proteins are dynamical entities organized in a network of correlated fluctuations whose changes can significantly affect binding at large distances [161].…”