Structural stability of E. coli transketolase to temperature and pH denaturation

Jahromi, R. Rahimi Fard; Morris, Phattaraporn; Martinez-Torres, R.J.; Dalby, Paul A.

doi:10.1016/j.jbiotec.2011.06.023

Cited by 17 publications

(30 citation statements)

References 49 publications

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“…This was likely to have resulted from partial loss of protein stability as expected when accumulating mutations for activity by random mutagenesis (Bloom et al, 2006), and whereby loss of stability can lead to loss of soluble expression in the cell (Calloni et al, 2005). Consistent with this link between stability and soluble expression, D469T gave 53% of the soluble expression level observed for wild type, and a decrease in the temperature at which aggregation was induced from 58 °C for wild type (Jahromi et al, 2011), to only 47 °C for D469T (Supplemental Experimental Procedures & Figure S3). By contrast, most of the double mutants were found to lose both activity and soluble expression levels much more markedly than the single mutants, suggesting that the loss of protein stability had crossed a critical threshold beyond which little of the protein was functionally folded.…”

Section: Resultssupporting

confidence: 57%

Directed evolution to re-adapt a co-evolved network within an enzyme

Strafford

Payongsri

Hibbert

et al. 2012

Journal of Biotechnology

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Section: Resultssupporting

confidence: 57%

Directed evolution to re-adapt a co-evolved network within an enzyme

Strafford

Payongsri

Hibbert

et al. 2012

Journal of Biotechnology

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“…Other solvent properties correlated even less with the molar holo-TK inactivation concentration, such as the molecular weight (R 2 =0.4), molecular volume (R 2 =0.4), topological polar surface area (R 2 =0.3), number of potential hydrogen bonds (R 2 =0.1), dipole moment (R 2 =0.2), and the dielectric constant of the co-solvent (R 2 =0.4). Although the Log(P) correlation was poor, it showed the highest R 2 which was consistent with previous observations that correlated hydrophobicity, and not the dielectric constant, to loss of enzyme activity and stability (Esakova et al, 2005;Miller et al, 2007;Galman et al, 2010;Jahromi et al, 2011;Strafford et al, 2012). However, various mechanisms have been used to explain such previous correlations, and so we sought to further characterise the solvent-induced inactivation of TK, as summarised in Table 3 and described below.…”

Section: Correlation Of Tk Activity To Calculated Organic Solvent Prosupporting

confidence: 81%

“…Several differences in the protein conformation of the holo and apo TK has been previously observed, for example the secondary and tertiary structure of holo-TK remained constant from pH 5 to 11, whereas the apo-TK secondary structure content increased with pH (Jahromi et al, 2011). Additionally, effects of varying cofactor concentrations on TK activity in holo-TK and apo-TK have been observed, which the latter showed inactivity in the lysate wild-type TK (Miller et al, 2007).…”

Section: Introductionmentioning

confidence: 75%

“…Fluorescence tryptophan spectroscopy has been previously successfully applied to provide tertiary structural information for TK (Esakova et al, 2005;Martinez-Torres et al, 2007;Aucamp et al 2008;Jahromi et al, 2011). The unfolding of E. coli TK can lead to both an increase or decrease in intrinsic fluorescence intensity, as observed previously where urea denaturation gave an initial increase followed by a decrease at higher urea concentrations (Martinez-Torres et al, 2007).…”

Section: Tertiary Structure Of Apo-tk and Holo-tk In Polar Co-solventsmentioning

confidence: 81%

“…While we have previously characterised the activity, thermostability and aggregation of transketolase at a wide range of pH, temperature and chemical denaturant concentrations (Martinez-Torres et al, 2007;Jahromi et al, 2011), its stability in a range of organic cosolvents has not been previously determined. TK unfolds irreversibly with urea, heat, or at low pH, and then aggregates, except in urea.…”

Section: Introductionmentioning

confidence: 99%

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Biophysical characterization of the inactivation ofE. colitransketolase by aqueous co-solvents

Morris

García-Arrazola

Rios‐Solis

et al. 2020

Preprint

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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 commonly used polar organic co-solvents: acetonitrile (MeCN), nbutanol (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 correlated to global conformational stability. The cosolvent 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.Aggregation was not found to be the driving mechanism of enzyme inactivation, but was in some cases an additional impact of solvent-induced local or global unfolding.TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) MeCN, or 6% (v/v) nBuOH over 3 hours. 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.

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Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties

et al. 2012

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Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTK gst ) retained 100% activity for one week at 50 8C and for 3 days at 65 8C, and has an optimum temperature range around 60-70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTK gst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTK gst from 1 L of culture. The reaction rate of mTK gst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTK gst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors -glycolaldehyde, d-glyceraldehyde and butyraldehyde -from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTK gst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.

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Structural stability of E. coli transketolase to temperature and pH denaturation

Cited by 17 publications

References 49 publications

Directed evolution to re-adapt a co-evolved network within an enzyme

Directed evolution to re-adapt a co-evolved network within an enzyme

Biophysical characterization of the inactivation ofE. colitransketolase by aqueous co-solvents

Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties

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