Abstract
Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.
Highlights
Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and aromatic substrates
The only exception was for holo-TK in 20% (v/v) AcCN, which induced global unfolding. This is consistent with previous studies that suggest enzyme activity loss in polar solvents is primarily due to the stripping of water from the protein surface and enzyme active-site, which in turn affects the active-site geometry, stability and catalytic function[73,74]
Local unfolding led to aggregation for holo-TK in certain solvents, but only after enzyme deactivation had already occurred
Summary
Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and aromatic substrates. This has led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. The mechanisms leading to partial inactivation of enzymes in organic solvents are still not clear, or generalizable to a wide range of enzymes such that they elucidate the relative roles of protein-solvent interactions, surface or active-site dehydration, structural denaturation, partial unfolding, subunit or cofactor dissociation, and enzyme a ggregation[30,31,32]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
