Abstract
Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins1,2,3,4. Nonetheless, its potential in even small proteins is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20100 combinations, which is beyond any existing experimental approach. In practice, selective modification of relatively few residues is sufficient for efficient improvement, functional enhancement and repurposing of existing proteins5. Moreover, computational methods have been developed to predict the locations and, in certain cases, identities of potentially productive mutations6,7,8,9. Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer a limited ability to identify beneficial mutations far from the active site, even though such changes may markedly improve the catalytic properties of an enzyme10. Machine learning methods have recently showed promise in predicting productive mutations11, but they frequently require large, high-quality training datasets, which are difficult to obtain in directed evolution experiments. Here we show that mutagenic hot spots in enzymes can be identified using NMR spectroscopy. In a proof-of-concept study, we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. The observed levels of catalytic efficiency exceed those of proteins designed using current approaches and are similar with those of natural enzymes for the reactions that they are evolved to catalyse. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to enable the full potential of directed enzyme evolution.
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Data availability
The crystallographic data and refinement statistics were deposited in the Protein Data Bank (PDB) with the entry code 7VUC (FerrElCat), 7VUR (AlleyCat9), 7VUS (AlleyCat9 with 6-NBT), 7VUT (AlleyCat10) and 7VUU (AlleyCat10 with 6-NBT). The NMR chemical shift data are provided as part of the Supplementary Information.
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Acknowledgements
This work was supported by the National Institutes of Health grant GM119634, the Japan Society for the Promotion of Science and the Alexander von Humboldt Foundation. The authors thank R. Fasan for the gift of the plasmid containing the Mb gene. J.R.H.T. thanks OpenEye Scientific Software.
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I.V.K., O.V.M. and A.N.V. designed the experiments. S.B., A.K., J.H.Y. and I.K. performed directed evolution, protein expression and characterization, and kinetic studies. A.D., A.N.V., O.V.M. and I.V.K. performed NMR titrations, backbone and side-chain assignment and NMR data analysis. Е.G.М., K.T. and J.R.H.T. expressed and crystallized the proteins and solved their structures. I.V.K. and O.V.M. wrote the manuscript with input from all the authors.
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Extended data figures and tables
Extended Data Fig. 1 Selection of negative controls for screening.
Left. Аll residues in myoglobin were sorted in bins based on their distance to the docked inhibitor (black) in FerrElCat. The residues in the van der Waals contact with the docked inhibitor were placed in bin 1 (red), the residues in direct contact with the residues in bin 1 were placed in bin 2 (orange), etc. A total of five bins were devised: red, orange, yellow, green and blue. Right. The list of the residues sorted in the five bins. Residues showing large backbone CSP and their immediate neighbors are highlighted in red and yellow, respectively. Unassigned positions and residues immediately next to unassigned stretches are shown in dark grey and light grey, respectively. Prolines are highlighted in blue. Residues showing small CSP that were selected as controls are either highlighted in green or labeled in green font (when located next to unassigned residues).
Extended Data Fig. 2 Substrate turnover by reduced Mb(L29I/H64G/V68A) (FerrElCat).
Reaction was monitored using stopped-flow at pH 8.0 for 30 s at 25 °C with 140 µM of 5-NBI and 5 nM of FerrElCat.
Extended Data Fig. 3 Catalytic parameters of Kemp eliminases evolved using directed evolution.
kcat/KM and kcat values for the evolved enzymes (top and bottom left, respectively) and improvement in kcat/KM and kcat achieved by directed evolution (top and bottom right, respectively). In cases where only kcat/KM was reported, we used 5 mM for KM, to obtain low estimate of the kcat.
Extended Data Fig. 4 Spectroelectrochemical determination of redox potentials of selected myoglobin mutants.
Mb(H64V) (left), Mb(H64G/V68A) (middle) and FerrElCat (right). The proteins were analyzed in 20 mM Tris-HCl, pH 8.0 at 20 °C in presence of the mediator (100 µM phenazine sulfate). The redox potentials (vs Ag/AgCl) are summarized in Supplementary Table 5. Data are presented as the mean values and the error bars represent standard deviations obtained from three independent measurements for Mb(H64V) and Mb(H64G/V68A). For FerrElCat, the experiment was repeated twice with essentially identical results, one set of data is shown.
Extended Data Fig. 5 Kemp elimination catalyzed by AlleyCat10 in presence of Ca2+ (black) and in absence of Ca2+ (red).
The activity of 0.1 µM AlleyCat10 was tested with 0.12-0.96 mM substrate in 20 mM HEPES, 100 mM NaCl, pH 7.0 at 22 °C with 10 mM CaCl2 (black) or 100 mM EDTA (red). Data are presented as the mean values and the error bars (most are small compared to the symbol size) represent standard deviations obtained from three independent measurements.
Extended Data Fig. 6 Absorbance spectra of Mb(H64V) (left) and Mb(L29I/H64G/V68A) (FerrElCat) (right) in the oxidized (black) and reduced (red) forms.
Spectra for the proteins (1.6 µM for Mb(H64V), 3.1 µM for FerrElCat) were collected in 20 mM HEPES, pH 7.0 in an anaerobic cuvette with a 1 cm pathlength at room temperature.
Extended Data Fig. 7 Michaelis-Menten plots of Kemp elimination catalyzed by reduced (unless otherwise stated) myoglobin mutants and by AlleyCat proteins.
Final reaction mixtures for myoglobin mutant analyses contained 1 mM L-ascorbic acid, 0.1 µM SOD, 20 nM catalase, 140-840 µM substrate, 1.5% acetonitrile in 20 mM Tris (pH 8.0). The protein concentration was 1 µM for Mb(H64V), 0.1 or 0.25 µM for Mb(H64V)-based double variants, 5 nM for Mb(H64G) or Mb(H64G)-based double or triple variants. For the AlleyCat proteins reaction mixtures contained 0.1 µM proteins with 0.12-0.96 mM substrate in 1.5% acetonitrile, 20 mM Tris, pH 8.0, 10 mM CaCl2, 100 mM NaCl. Kinetic parameters are summarized in Table 1 and Extended Data Table 1. Data are presented as the mean values and the error bars represent standard deviations obtained from at least three independent measurements (six for Mb(H64G) and reduced Mb(H64V).
Supplementary information
Supplementary Data 1
This file contains Supplementary Figs. 1–11 and Tables 1–6.
Supplementary Data 2
The file contains the chemical shifts values, CSP values and Z-scores for individual residues derived from NMR titrations.
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Bhattacharya, S., Margheritis, E.G., Takahashi, K. et al. NMR-guided directed evolution. Nature 610, 389–393 (2022). https://doi.org/10.1038/s41586-022-05278-9
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DOI: https://doi.org/10.1038/s41586-022-05278-9
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