Using Enzymes to Tame Nitrogen-Centered Radicals for Enantioselective Hydroamination

The construction of C–N bonds is essential for the preparation of numerous molecules critical to modern society1,2. Nature has evolved enzymes to facilitate these transformations using nucleophilic and nitrene transfer mechanisms3,4. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centered radicals, which serve as valuable species for C–N bond formation. Herein, we describe a platform for generating nitrogen-centered radicals within protein active sites, thus enabling asymmetric hydroamination reactions. Using flavin- dependent ‘ene’-reductases with an exogenous photoredox catalyst, amidyl radicals are generated selectively within the protein active site. Empowered by directed evolution, these enzymes are engineered to catalyze 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies suggest that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlight the opportunity for synergistic catalyst strategies to unlock new enzymatic functions.

Partial synthetic models of FeMoco with sulfide and carbyne ligands: Effect of interstitial atom in nitrogenase active site

Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe7S8C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe3S3C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M’Fe3S3X]n−, where M’ = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M’ results in minor changes, the chelating, μ3-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.

Protein active site prediction for early drug discovery and designing

Adenosine triphosphate (ATP) is an energy compound present in living organisms and is required by living cells for performing operations such as replication, molecules transportation, chemical synthesis, etc. ATP connects with living cells through specialized sites called ATP-sites. ATP-sites are present in various proteins of a living cell. The life span of a cell can be controlled by controlling ATP compounds and without the provision of energy to ATP compounds, cells cannot survive. Countless diseases treatment (such as cancer, diabetes) can be possible once protein active sites are predicted. Considering the need for an algorithm that predicts ATP-sites with higher accuracy and effectiveness, this research work predicts protein ATP sites in a very novel way. Till now Position-specific scoring matrix (PSSM) along with many physicochemical properties have been used as features with deep neural networks in order to create a model that predicts the ATP-sites. To overcome this problem of complex computation, this exertion proposes k-mer feature vectors with simple machine learning (ML) models to attain the same or even better performance with less computation required. Using 2-mer as feature vectors, this research work trained and tested five different models including KNN, Conv1D, XGBoost, SVM and Random Forest. SVM gave the best performance on k-mer features. The accuracy of the created model is 96%, MCC 90% and ROC-AUC is 99%, which are the same or even better in some aspects than the state-of-the-art results. The state-of-the-art results have an accuracy of 97%, MCC 78% and ROC-AUC is 92%. One of the benefits of the created model is that it is much simpler and more accurate.

Cluster Models of FeMoco with Sulfide and Carbyne Ligands: Effect of Interstitial Atom in Nitrogenase Active Site

<p>Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an iron-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMoco displays eight metal centers bridged by sulfides and a carbide having the MoFe₇S₈C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing its function, we isolated synthetic models of subsite MFe₃S₃C displaying sulfides and a carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*MFe₃S₃X]^n-, where M = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M results in minor changes, the μ₃-bridging ligand X has a large impact on reduction potentials, with differences higher than 1 V, even for the same formal charge, the most reducing clusters being supported by the carbyne ligand. </p>

Cluster Models of FeMoco with Sulfide and Carbyne Ligands: Effect of Interstitial Atom in Nitrogenase Active Site

<p>Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an iron-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMoco displays eight metal centers bridged by sulfides and a carbide having the MoFe₇S₈C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing its function, we isolated synthetic models of subsite MFe₃S₃C displaying sulfides and a carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*MFe₃S₃X]^n-, where M = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M results in minor changes, the μ₃-bridging ligand X has a large impact on reduction potentials, with differences higher than 1 V, even for the same formal charge, the most reducing clusters being supported by the carbyne ligand. </p>

Quantification of Electric Field inside the Protein Active Sites and Fullerenes

While electrostatic interactions are exceedingly accountable for biological functions, no simple method exists to directly estimate or measure the electrostatic field in the protein active sites. The electrostatic field inside...

The Role of Electrostatics in Enzymes: Do Biomolecular Force Fields Reflect Protein Electric Fields?

<div><div><div><p>Preorganization of large, directionally oriented, electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanism in many enzymes, and may be efficiently investigated at the atomistic level with molecular dynamics simulations. Here we evaluate the ability of the AMOEBA polarizable force field, as well as the additive Amber ff14SB and Charmm C36m models, to describe the electric fields present inside the active site of the peptidyl-prolyl isomerase cyclophilin A. We compare the molecular mechanical electric fields to those calculated with a fully first principles quantum mechanical (QM) representation of the protein, solvent, and ions, and find that AMOEBA consistently shows far greater correlation with the QM electric fields than either of the additive force fields tested. Catalytically-relevant fields calculated with AMOEBA were typically smaller than those observed with additive potentials, but were generally consistent with an electrostatically-driven mechanism for catalysis. Our results highlight the accuracy and the potential advantages of using polarizable force fields in systems where accurate electrostatics may be crucial for providing mechanistic insights.</p></div></div></div>

The Role of Electrostatics in Enzymes: Do Biomolecular Force Fields Reflect Protein Electric Fields?

<div><div><div><p>Preorganization of large, directionally oriented, electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanism in many enzymes, and may be efficiently investigated at the atomistic level with molecular dynamics simulations. Here we evaluate the ability of the AMOEBA polarizable force field, as well as the additive Amber ff14SB and Charmm C36m models, to describe the electric fields present inside the active site of the peptidyl-prolyl isomerase cyclophilin A. We compare the molecular mechanical electric fields to those calculated with a fully first principles quantum mechanical (QM) representation of the protein, solvent, and ions, and find that AMOEBA consistently shows far greater correlation with the QM electric fields than either of the additive force fields tested. Catalytically-relevant fields calculated with AMOEBA were typically smaller than those observed with additive potentials, but were generally consistent with an electrostatically-driven mechanism for catalysis. Our results highlight the accuracy and the potential advantages of using polarizable force fields in systems where accurate electrostatics may be crucial for providing mechanistic insights.</p></div></div></div>

A nearly on-axis spectroscopic system for simultaneously measuring UV–visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline

UV–visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probedviacaged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.