Stabilization of Hyperactive Dihydrofolate Reductase by Cyanocysteine-mediated Backbone Cyclization

Abstract

Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine-and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteinemediated cyclization method is complementary to the inteinmediated cyclization method in stabilizing a protein without affecting its activity.

Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

“…It has been reported that, in case of the spatial proximity of flexible protein termini, the covalent linkage of the termini may have no or little effect on the overall structure of the protein. Therefore, enzymes modified in this way may benefit from enhanced stability with no reduction in enzymatic activity [21,34]. Accordingly, an inteinmediated cyclization technique was selected for the enhancement of srtA stability.…”

“…Additionally, the renaturation rates for these circularly-closed protein molecules are greatly increased [20,21]. The structure of the recombinantly expressed srtA catalytic domain has been well studied, and the spatial proximity of the protein termini has been demonstrated [12,22].…”

Triterpenoid Biosynthesis and Engineering in Plants

“…In Solanaceae , CYP71Ds are involved in sesquiterpenoid phytoalexin biosynthesis. Protein engineering of CYP71Ds based on sequence alignment analysis with phylogenetically related P450 species and homology modeling successfully enhanced the catalytic efficiencies of the enzymes (Takahashi et al, 2007). In Glycyrrhiza , not only glycyrrhizin-producing species but also non-producing species can produce other oleanane-type triterpenoid saponins (Hayashi et al, 2000).…”

How Protein Stability and New Functions Trade Off

“…Such observations (notwithstanding exceptions such as residues that contribute to both function and stability [24]–[26], and cases where enzyme stability can be increased without comprising function [10], [27]–[31]) led to the generally accepted principle of stability-function tradeoffs [16],[19] that was later extended to tradeoffs between new functions and stability [32]. However, as discussed below, we surmise that there exists a fundamental difference between mutations in key catalytic residues that relate to the well established stability-function tradeoff, and mutations that mediate the evolutionary divergence of new functions.…”