Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology
Abstract
:1. Introduction
2. Results and Discussion
2.1. Kinetic Characterization of TS from Salmonella typhimurium
2.2. Identification and Characterization of An Efficient Position for Light-Activation of TS
2.3. Inhibition of Catalytic Actvity of TS(aL58ONBY) Can Be Reversed by Light
2.4. Allosteric Activation of TrpA and TrpB is Inhibited by aL58ONBY
2.5. Optimized Light-Activation of TS by Combination of Kinetic Effects
4. Materials and Methods
4.1. Strains, Enzymes, and Chemicals
4.2. Subcloning of the trpA, ectrpA, ectrpB, and vioA Genes
4.3. Site-Directed Mutagenesis of trpA and trpB
4.4. Expression and Purification of TS
4.5. Expression and Purification of Auxiliary Enzymes
4.6. Activity Measurements
4.7. Tryptic Digest and MS Analysis of Protein Sequences
4.8. MD Simulation
4.9. Generating a Sequence Logo for TrpA Subunits
4.10. Native MS Analysis
4.11. CD Analysis
4.12. Analytical Size-Exclusion Chromatography
4.13. UV/Vis Spectral Analysis of Serine Binding
4.14. Equations
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
UAA | Unnatural amino acid |
aaRS | Aminoacyl-tRNA synthetase |
ONBY | o-nitrobenzyl-O-tyrosine |
LAF | Light activation factor |
TS | Tryptophan synthase from Salmonella typhimurium |
IGP | Indole glycerol phosphate |
GAP | Glyceraldehyde-3-phosphate |
PLP | Pyridoxal phosphate |
AA | Aminoacrylate |
IA | Internal aldimine |
TS | Tryptophan synthase from Saccharomyces cerevisiae |
ecTS | Tryptophan synthase from Escherichia coli |
HRP | Horse-radish peroxidase |
MS | Mass spectrometry |
MD | Molecular dynamics |
CD | Circular dichroism |
ai | “as isolated” |
hν | Irradiated |
SEM | Standard error of mean |
SE | Standard error |
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Reaction | Parameter | Dimension | Value TS | Value ecTS |
---|---|---|---|---|
TrpA 1 | kcat | [min−1] | 2.4 ± 0.2 | 1.5 ± 0.1 |
KmIGP | [µM] | 29.4 ± 8.4 | 17.6 ± 5.4 | |
kcat/KmIGP | [s−1M−1] | 13.6 · 102 | 14.2 · 102 | |
TrpB 2 | kcat | [min−1] | 24.1 ± 0.6 | 21.8 ± 0.7 |
Kmindole | [µM] | 51.0 ± 4.5 | 10.6 ± 1.6 | |
kcat/Kmindole | [s−1M−1] | 7.9 · 103 | 34.3 · 103 | |
KmSer | [mM] | 0.53 ± 0.05 | 0.52 ± 0.07 | |
kcat/KmSer | [s−1M−1] | 7.6 · 102 | 7.0 · 102 | |
TS 3 | kcat | [min−1] | 21.3 ± 0.6 | 28.3 ± 1.2 |
KmIGP | [µM] | 25.9 ± 2.6 | 24.9 ± 3.7 | |
kcat/KmIGP | [s−1M−1] | 13.7 · 103 | 18.9 · 103 | |
KmSer | [mM] | 0.23 ± 0.03 | 0.35 ± 0.05 | |
kcat/KmSer | [s−1M−1] | 15.4 · 102 | 13.5 · 102 |
Reaction | Parameter | Dimension | Value TS(aL58ONBYai) | LAF | Value TS(aL58ONBYhν) | Value TS(L58Y) |
---|---|---|---|---|---|---|
TrpA | kcat | [min−1] | 0.19 ± 0.02 | 8 → | 1.50 ± 0.07 | 2.30 ± 0.09 |
KmIGP | [µM] | 549 ± 92 | 3 ← | 194 ± 23 | 234 ± 20 | |
kcat/KmIGP | [s−1M−1] | 0.06 · 102 | 22 → | 1.29 · 102 | 1.64 · 102 | |
TrpB | kcat | [min−1] | 0.9 ± 0.0 | 13 → | 12.1 ± 0.4 | 16.3 ± 0.3 |
Kmindole | [µM] | 3.4 ± 0.4 | 4 → | 13.9 ± 1.5 | 37.1 ± 2.4 | |
kcat/Kmindole | [s−1M−1] | 4.41 · 103 | 3 → | 14.51 · 103 | 7.32 · 103 | |
KmSer | [mM] | 3.9 ± 0.3 | 6 ← | 0.7 ± 0.1 | 0.7 ± 0.0 | |
kcat/KmSer | [s−1M−1] | 0.04 · 102 | 72 → | 2.88 · 102 | 3.88 · 102 | |
TS | kcat | [min−1] | 0.3 ± 0.0 | 18 → | 5.4 ± 0.2 | 10.9 ± 0.5 |
KmIGP | [µM] | 103 ± 18 | 2 ← | 58 ± 10 | 29 ± 6 | |
kcat/KmIGP | [s−1M−1] | 0.05 · 103 | 31 → | 1.55 · 103 | 6.26 · 103 | |
KmSer | [mM] | 0.6 ± 0.1 | 3 ← | 0.2 ± 0.0 | 0.2 ± 0.0 | |
kcat/KmSer | [s−1M−1] | 0.08 · 102 | 56 → | 4.50 · 102 | 9.08 · 102 |
Reaction | Parameter | Dimension | Value TS(aL58ONBY)ai | Value TS(aL58Y) |
---|---|---|---|---|
TrpA (−Ser) | V01 | [µM min−1] | 0.09 ± 0.01 | 0.16 ± 0.01 |
Vmax2 | [µM min−1] | 0.12 ± 0.00 | 1.86 ± 0.06 | |
Vmax/V03 | 1 | 12 | ||
TrpA (+Ser) | V0 | [µM min−1] | 0.02 ± 0.00 | 0.03 ± 0.04 |
Vmax | [µM min−1] | 0.09 ± 0.00 | 4.80 ± 0.33 | |
Vmax/V0 | 4 | 160 |
Reaction | Parameter | Dimension | Value TS(aL58ONBYai) | Value TS(aL58ONBY) | Value TS(aL58Y) |
---|---|---|---|---|---|
TrpA (−Ser) | V01 | [min−1] | 0.15 | 0.17 | 1.27 |
Vhν2 | [min−1] | — | 0.73 | 0.84 | |
LAF | — | 4 | 0.66 | ||
TrpA (+Ser) | V0 | [min−1] | 0.20 | 0.20 | 17.83 |
Vhν | [min−1] | — | 8.46 | 10.01 | |
LAF | — | 42 | 0.56 | ||
TS | V0 | [min−1] | <0.03 | <0.02 | 4.95 |
Vhν | [min−1] | — | 2.83 | 3.10 | |
LAF | — | >141 | 0.63 |
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Kneuttinger, A.C.; Zwisele, S.; Straub, K.; Bruckmann, A.; Busch, F.; Kinateder, T.; Gaim, B.; Wysocki, V.H.; Merkl, R.; Sterner, R. Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology. Int. J. Mol. Sci. 2019, 20, 5106. https://doi.org/10.3390/ijms20205106
Kneuttinger AC, Zwisele S, Straub K, Bruckmann A, Busch F, Kinateder T, Gaim B, Wysocki VH, Merkl R, Sterner R. Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology. International Journal of Molecular Sciences. 2019; 20(20):5106. https://doi.org/10.3390/ijms20205106
Chicago/Turabian StyleKneuttinger, Andrea C., Stefanie Zwisele, Kristina Straub, Astrid Bruckmann, Florian Busch, Thomas Kinateder, Barbara Gaim, Vicki H. Wysocki, Rainer Merkl, and Reinhard Sterner. 2019. "Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology" International Journal of Molecular Sciences 20, no. 20: 5106. https://doi.org/10.3390/ijms20205106