Targeted mutagenesis of a thermophilic mevalonate kinase for efficient catalysis at lower temperatures.

Abstract

Thermophile-derived enzymes are of interest for cell-free biocatalysis due to their intrinsic thermostability, which simplifies purification via heat treatment and correlates with enhanced activity retention under cell-free reaction conditions. However, thermal stability often comes at the expense of slower turnover rates for reactions that need to be conducted at ambient temperature out of concern for cofactor stability and to reduce energy requirements. This study demonstrates a targeted engineering strategy to increase the room temperature activity of the thermophilic mevalonate kinase from Methanocaldococcus jannaschii. Site-saturation mutagenesis of evolutionarily variable residues within the mevalonate binding pocket generated numerous mutants with increased activity at 22°C. Fifteen mutants demonstrated a two- to four-fold increase in activity, and the most active mutant, I10S, achieved a five-fold increase in kcat relative to the wild-type. Importantly, the I10S mutant retained thermostability comparable to the wild-type, and a minimal decline in long-term stability was observed. This mutagenesis strategy may have broader applications towards tuning thermophilic enzyme activity at desired operating temperatures to enhance the efficiency of cell-free reactions while reducing operating costs.