Abstract
The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity-stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.
Highlights
The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability
Saturation mutagenesis of two TK active-site residues within D469T/R520Q led to several variants, including S385Y/ D469T/R520Q (3M), that were active on three benzaldehyde derivatives, in contrast to WT TK, which was active only on nonaromatic aldehydes [15, 16]
Directed evolution toward 3M shifted the substrate specificity toward previously unaccepted aromatic aldehydes, and increased its promiscuity compared with WT TK
Summary
The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. During the process of directed evolution for improved activity or substrate specificity, stability loss is commonly observed when mutations are accumulated primarily for function [1, 2]. This negative correlation between enzyme stability and activity, the so-called activity–stability trade-off, has been well documented [3,4,5]. To increase potential use for the synthesis of novel dihydroxy-ketone compounds, E. coli TK has been engineered by a series of smartlibrary approaches to expand its substrate scope from phosphorylated to nonphosphorylated polar acceptors, to nonpolar aliphatic substrates, and on to hetero-aromatic and nonpolar aromatic substrates, which makes it a great model for investigation of the relationship between stability and new functions, when using guided or semirational-directed evolution strategies. The kinetic analysis of 3M showed that this variant improved the activities toward
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