Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents.

Autor: Morris P; Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London, WC1E 6BT, UK.; Chemical Metrology and Biometry Department, National Institute of Metrology, 3/4-5 Moo 3, Klong 5, Klong Luang, 12120, Pathumthani, Thailand., García-Arrazola R; Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London, WC1E 6BT, UK., Rios-Solis L; Institute for Bioengineering, School of Engineering, University of Edinburgh, Edinburgh, EH9 3JL, UK.; Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, King's Buildings, Edinburgh, EH9 3JL, UK., Dalby PA; Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London, WC1E 6BT, UK. p.dalby@ucl.ac.uk.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2021 Dec 08; Vol. 11 (1), pp. 23584. Date of Electronic Publication: 2021 Dec 08.
DOI: 10.1038/s41598-021-03001-8
Abstrakt: 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.
(© 2021. The Author(s).)
Databáze: MEDLINE
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