Heteromeric enzyme complexes, which accelerate a tremendous amount of biological reactions, are often allosterically regulated by their protein partners, being the isolated catalytic subunits less active.1,2 Stand-alone enzyme subunits, however, are desirable for biosynthetic applications as lower metabolic loads on the host cell are needed, and the efforts to engineer activity, substrate specificity, etc. are drastically simplified. As a result, several efforts have been made to engineer the isolated subunits to function independently and, despite recent advances in the field,3,4 major challenges remain to be addressed. The understanding of the structural and mechanistic basis of beneficial random mutations remains elusive, in particular, how mutations far from the active site affect the dynamic behavior of proteins and confer improved catalytic properties in the absence of their protein-binding partners. Here, we applied microsecond MD simulations together with Markov models5 to the well-characterized LovD wild-type enzyme and laboratory-evolved variants,3 to analyze the effect of distant mutations on the allosteric regulation of heteromeric enzymes and their catalytic activities. Here, we demonstrate how this approach allows us to dissect dynamic data into a number of structural features that are beneficial for catalysis, preactivating the catalytic residues towards an active conformation, and obviating the need of allosteric regulation for efficient catalysis.

Molecular dynamics simulations and Markov models of natural and evolved stand-alone LovD enzyme variants

254th National Meeting (2017)

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