Clock-Turning Control of P450 TxtE Regioselectivity Enables Precise Site Functionalization of Aromatic Compounds

Abstract

Aromatic nitration is a key transformation in the synthesis of pharmaceuticals, functional materials, and fine chemicals, yet selective functionalization of electronically deactivated sites remains a formidable challenge. The C6 position of l-tryptophan is particularly inert due to the high conjugation and low electron density of the indole ring, and no chemical or enzymatic system has achieved efficient single-step nitration at this site. Here we report a computer-aided regioselective design platform that precisely reprograms cytochrome P450 TxtE to enable C6-selective nitration. Guided by MD simulations, MM/PBSA energy decomposition, and QM/MM calculations, we identified key residues that control substrate orientation and engineered the H176F/A248F double mutant, which achieves >99% C6 selectivity with the highest total turnover number among all variants. High-resolution crystallographic analysis (1.53 Å) combined with QM/MM calculations revealed that, in the H176FA248F, steric repacking of the active pocket and substrate flipping lower the C6 nitration barrier from 54.3 kcal·mol^–1 in the WT to 17.9 kcal·mol^–1. This work not only endows TxtE with unprecedented C6-selective nitration capability, but also reveals that the clock-like control of P450 TxtE regioselectivity enables precise site-selective functionalization of aromatic compounds, providing a new conceptual framework and a generalizable strategy for targeted functionalization of aromatic scaffolds.

1. Introduction

Owing to its two electron-deficient oxygen atoms and a partially positively charged nitrogen atom, nitro groups exhibit a strong electron-withdrawing effect. , When attached to an aromatic ring, they reduce the electron density of the ring through conjugative effects, thereby modulating its reactivity and regioselectivity and promoting electrophilic substitution at specific positions. Based on this electronic effect, numerous nitroaromatic compounds have been developed for applications in pharmaceuticals, agrochemicals, dyes, and functional materials, − making them one of the most widely used industrial chemicals. However, naturally occurring nitroaromatic compounds are relatively rare, therefore the vast majority of those used in industry are produced synthetically. Classical electrophilic aromatic substitution uses mixed acid systems (concentrated H₂SO₄ and HNO₃) and remains the predominant strategy for the synthesis of nitroaromatics. However, this method requires harsh reaction conditions and generally fails to achieve high regioselectivity at the electron-deficient positions of the aromatic rings, , which limits its applicability in the synthesis of complex, functionalized molecules. Recently, alternative nitration strategies have been developed. For example, Parac-Vogt et al. reported a lanthanide-based catalytic system for toluene nitration, highlighting the potential of nonconventional Lewis acid systems in aromatic nitration, while Kumar et al. employed solid acid catalysts as substitutes for strong traditional liquid acids to reduce corrosiveness and improve operational safety. Although these new approaches offer advantages in terms of green chemistry, achieving precise control through regioselectivity remains a major challenge, particularly for that of electron-deficient positions on aromatic rings.

Enzymatic catalysis has emerged as an attractive strategy for aromatic nitration because of its mild reaction conditions, excellent regioselectivity, and engineering flexibility. , In 2012, Sarah et al. were the first to discover that cytochrome P450 TxtE, extracted from Streptomyces scabies, catalyzes the highly selective nitration of L-tryptophan at the C4 position, demonstrating the potential of biocatalysis for selective aromatic nitration. Building on this discovery, Arnold et al. identified that the His176 residue located in the F/G loop region of TxtE plays a pivotal role in regioselectivity control; its mutation of phenylalanine (H176F) redirects the nitration site from C4 to C5. This sensitivity aligns with the broader catalytic paradigm of cytochrome P450 enzymes, which activate molecular oxygen at the heme cofactor and rely on substrate orientation to dictate regio-outcome. − However, no enzymatic system has been reported that enables highly selective nitration at the C6 position in a single step, highlighting a fundamental limitation of both chemical and enzymatic nitration strategies to date. This is likely due to the fact that the C6 site resides in the highly conjugated indole ring system where the electron density is relatively low, which protects the site from electrophilic attacks. , Notably, 6-nitrotryptophan possesses unique optical properties, such as fluorescence quenching and UV absorption shifts, that make it a valuable probe for studying protein conformational changes. Therefore, the development of an efficient enzymatic strategy capable of achieving regioselective nitration at the C6 position of tryptophan is of scientific and practical importance.

In this study, we combined computer-aided design with structural analysis to identify key residues that control substrate orientation and engineered the H176F/A248F double mutant, which for the first time achieves >99% selectivity for C6 nitration. This achievement has overcome the long-standing challenge of activating the C6 site in conventional aromatic nitration and highlights the potential of this enzyme for the synthesis of aromatic nitro compounds. By integrating high-resolution crystal structure determination, energy profiling, molecular interaction analysis, and QM/MM pathway tracing, we systematically elucidated the molecular mechanism by which the enzyme confers C6 regioselectivity. Compared with traditional nitration reactions and recently developed chemical catalytic strategies, this work provides a theoretical foundation for green and precise aromatic nitration and opens new avenues for the future design of highly selective and efficient biocatalytic systems.

2. Results and Discussion

2.1. Challenges and Conformational Constraints of C6 Nitration of Tryptophan

To achieve highly selective nitration at the C6 position of tryptophan, we first performed QM calculations to investigate the classical NO₂ ⁺ electrophilic substitution on the indole ring of tryptophan in the absence of enzymatic catalysis, with the aim of systematically evaluating the intrinsic nitration reactivity of different positions on the aromatic ring. As shown in Figure A, the predicted order of electrophilic reactivity is C5 > C7 > C4 > C6, with the C6 position being the least reactive, indicating that this site is intrinsically unfavored under nonenzymatic conditions. Notably, the calculated energy barrier for the transition state at C6 is only 0.43 kcal·mol^–1 higher than that at C4, however, wild-type TxtE experimentally exhibits strong preference for C4 nitration. This discrepancy suggests that the microenvironment of the enzyme active pocket plays a decisive role in governing regioselectivity. ,

1.

Computational analysis of tryptophan nitration reactivity and TxtE selectivity challenge. (A) QM-calculated (Single-point energies were then computed at the B3LYP/def2-TZVP level with Grimme’s D3 dispersion corrections) relative reactivity of indole-ring carbons toward electrophilic nitration. (B) Probability distribution of the shortest distances between peroxynitrite N and indole carbon atoms, as obtained from molecular dynamics simulations and docking studies. (C) RMSD of apo-TxtE and population of C6-oriented conformations. (D) Representative C6- and C4-oriented conformations selected as initial MD structures. (E) Binding free energies of C6- vs C4-oriented states.

To validate this hypothesis and investigate why wild-type TxtE fails to catalyze C6 nitration, the ferric peroxynitrite species was first positioned at the heme active site of apo-TxtE (PDB ID: 4TPN), followed by a 100 ns molecular dynamics simulation. Subsequently, snapshots were extracted from the MD trajectory, and tryptophan was docked into each snapshot. The resulting configurations were then used to analyze the distribution of distances between the nitrogen atom of peroxynitrite and the four aromatic carbons of tryptophan (C4, C5, C6, and C7) was analyzed (Figure B). The results indicate that C4 and C7 were the most accessible positions, with C4 being the closest site in 59% of the sampled conformations, whereas C5 and C6 account for only 5.91%. This conformational distribution was consistent with the experimentally observed preference of TxtE for C4 nitration. However, these results only demonstrate the geometric feasibility of a radical attack at C4 and do not reveal the energetic stability of the sampled conformations. Further MD analysis showed that the wild-type enzyme reached a stable state after approximately 70 ns (Figure C), with the C6-oriented conformations arising primarily during this period, where the C6 position of the phenyl ring is closest to the nitrogen atom. Free energy calculations further revealed that the global minimum free-energy state was reached at 83 ns (Figure S19), corresponding to a conformation where C6 is the closest site, with the distance between C6 and the nitrogen atom of peroxynitrite being 3.4 Å (Figure D). This finding suggests that TxtE possesses intrinsic potential to form C6-oriented conformations, despite its C4 selectivity in our earlier experiments.

To evaluate the catalytic potential of the C6-oriented conformation, we selected this conformation as the starting structure for MD simulations, using the optimal C4-oriented conformation as a control (Figure D). The MMPBSA results showed that the binding free energy of the C6-oriented conformation was 59.33 kcal·mol^–1 higher than that of the C4-oriented conformation (Figure E), indicating that C6 binding was thermodynamically unfavorable. This finding explained the inability of TxtE to catalyze C6 nitration. Nevertheless, the simulations also showed that 5.91% of the conformations place the C5 and C6 position close to peroxynitrite. Since C5 nitration has been experimentally observed, this indicates that TxtE does not fully exclude the C6 site. Therefore, we hypothesize that introducing mutations to adjust the active-site volume or hydrogen-bonding network would induce a substrate conformational flip and stabilize C6-oriented binding, thereby enabling a regioselectivity switch.

2.2. Conformational Reorientation Enables C6 Nitration by TxtE

Based on this hypothesis, We focused on mutating key residues in the substrate-binding pocket that govern the orientation of the indole ring, with the aim of reshaping the local steric environment to better accommodate a C6-oriented binding mode of l-tryptophan. Arnold et al. reported that M88 and F395 in TxtE form a hydrophobic clamp (Figure A) that precisely locks to the indole plane of the substrate. Considering that the hydrophobic interaction between F395 and the substrate (Figure A) is essential for maintaining catalytic activity, excessive modifications at this position may abolish catalytic ability. Therefore, we performed saturation mutagenesis at M88 to explore whether local conformational perturbations promote C6 nitration (Figure A). Systematic screening of the resulting variants, combined with retention time analysis and characteristic UV–vis absorption spectra, enabled the assignment of the nitration site (Figure S1). Most M88 variants completely lost activity; however, unexpectedly, M88Y not only retained catalytic activity but also produced the desired 6-nitrotryptophan, albeit accompanied by a substantial amount of C4-nitrated product (Table ). This was the first experimental evidence that TxtE induced C6 nitration via site-directed mutagenesis. To improve the selectivity of TxtE toward 6-nitrotryptophan, we analyzed the mode of binding of the M88Y variant to the substrate. After mutating M88 to Y in apo-WT, we performed docking of the resulting variant with the substrate. The results showed that the M88Y substitution disrupted the native hydrogen-bonding network and stabilized substrate binding in the wild-type, in which Trp formed hydrogen bonds with T296 and R59. In the mutant, these interactions shifted mainly to N293, while new hydrophobic contacts with I244 and M173 formed, driving the conformational reorientation of the substrate within the active pocket (Figure B). We hypothesized that this conformational rearrangement underlies the ability of M88Y to catalyze C6 nitration. Based on this finding, we introduced combinatorial mutations at residues within 4 Å of the substrate (M173 and I244) in the M88Y background to enhance C6 selectivity. However, all the double mutants were catalytically inactive, which suggesting that although M88Y provides a basis for selectivity control, its intrinsically low catalytic activity (total turnover number, TTN = 3.7, Table ) makes further mutagenesis highly likely to induce destabilization of the protein conformation, ultimately leading to activity loss.

2.

Structural comparison of TxtE substrate-binding pockets before and after M88Y mutation. (A) In WT (PDB ID: 4TPO), Met88 and Phe395 form a hydrophobic clamp that precisely locks the indole plane of l-tryptophan, stabilized by hydrogen bonds with T296 and R59. (B) In M88Y, substrate forms a new H-bond with N293 and additional hydrophobic contacts with I244 and M173. Purple dashed lines indicate hydrophobic interactions; green dashed lines, hydrogen bonds.

1. l-tryptophan Nitration Catalyzed by Different Variants .

entry	enzyme	product	TTN	selectivity
1	M88 others	n.r	-	-
2	M88Y	4,6	3.7	83:19
3	M88YI244NNK	n.r	-	-
4	M88YM173NNK	n.r	-	-
5	A248S	4	5.4	>99
6	A248S others	n.r	-	-

^aReactions contained 10 μM purified enzyme, 1 mM l-tryptophan, 2 mM NOC-5, 2 mM NADP⁺, 10 mM glucose, and 25 U GDH in 25 mM Tris–HCl (pH 8.0) at 20 °C for 12 h (300 μL total volume). n.r. = no reaction. 4 = 4-nitrotryptophan, 6 = 6-nitrotryptophan, and NNK indicates a saturated mutation.

Next, we shifted our focus to another residue, A248, located within the hydrophobic pocket prior to the mutation (Figure A) and performed saturation mutagenesis. Among the variants, only A248S retained its catalytic activity, exhibiting regioselectivity comparable to that of the wild-type (Table ). Taken together, these results suggested that M88Y and A248S serve as key residues for biasing the substrate conformation toward the C6 position. Mutagenesis designs based solely on static crystal structures have inherent limitations because excessive perturbation of the catalytic core can abolish enzyme activity. Considering that enzymes undergo highly dynamic conformational changes during the catalytic cycle, − we hypothesize that a systematic analysis of the dynamic behavior of the enzyme–substrate complex using molecular dynamics and related computational methods could provide structural guidance for rational mutagenesis, offering a promising approach to enhance regioselectivity for C6 nitration of tryptophan.

2.3. A Computer-Aided Platform for Directed Regioselectivity Drives C6 Nitration

Based on the aforementioned findings, we constructed a computer-aided platform for directed regioselective design (Figure A). This platform integrates substrate-binding conformations, constrained MD simulations, and energy decomposition analysis to systematically identify key residues that influence substrate positioning, which are then subjected to combinatorial mutagenesis in conjunction with experimental validation to precisely remodel the nitration site. To assess the effectiveness of the platform, we first determined the primary binding mode of the substrate in the active pocket using molecular docking analysis. Considering that the spatial distance between the nitrogen atom of the Fe (III)–OONO intermediate and different positions on the aromatic ring is a critical determinant of nitration site selectivity, we introduced spatial restraints in MD simulations to maintain the C6 carbon of the substrate indole ring within 4 Å of the nitrogen atom of peroxynitrite, ensuring adequate sampling of the C6-oriented binding conformations. Then, constrained MD trajectories were analyzed using MM/PBSA energy decomposition to identify the residues that negatively affected the stability of the C6-oriented conformation. This analysis revealed a set of residues, including H176, that destabilized C6-oriented binding (Figure B,C). Arnold et al. reported that H176F/Y/W mutations significantly alter regioselectivity by redirecting the product from C4 to C5. Based on this, we introduced the H176Y/F/W mutation into the A248S background. The results showed that A248S/H176F exhibited the same product profile as the H176F single mutant, retaining high selectivity for C5 (Figure D). In contrast, both the A248S/H176Y and A248S/H176W mutants exhibited detectable C6 nitration, although they were accompanied by a higher proportion of regioisomers. Among these, A248S/H176W generated C5 and C6 products with a C6 selectivity of 41%. Comparatively, A248S/H176Y yielded products at C4, C5, and C6 but exhibited higher C6 selectivity (67%) and superior catalytic efficiency (Figure E). Consequently, we selected A248S/H176Y as the new conformational platform and attempted to combine it with a previously validated M88Y mutation capable of producing C6 nitration, with the aim of further enhancing C6 selectivity. The regioselectivity was distributed between C5 and C6 (Figure D), indicating a further trend toward regioselective redirection, although C6 selectivity decreased by 9% relative to that of the prior mutant (Figure D). To systematically explore the role of M88 in regioselectivity, we performed saturation mutagenesis of M88 in an A248S/H176Y background, excluding Tyr. All resulting variants were inactive, consistent with the results of previous attempts of single-site saturation mutagenesis at M88. These findings indicate that M88 is a critical residue for substrate recognition, tolerating only Tyr substitutions, and that the M88Y mutation can significantly influence substrate orientation.

3.

Computer-aided regioselective rational design platform and results. (A) Overview of the computational–experimental workflow. (B) Heat map of MMPBSA energy decomposition. (C) Residues disfavoring C6 orientation in constrained MD (including H176 and Y89). (D) Product distribution of TxtE variants. (E) Catalytic efficiencies of selected mutants. (F) Pocket volume analysis from CASTpFold.

After establishing the critical role of M88 in substrate recognition, we focused on another class of residues that might influence substrate-binding stability. Arnold et al. reported that Y89 in TxtE can form a stable hydrogen-bonding network with the substrate via its phenolic hydroxyl group, thereby locking the binding conformation of the substrate. Consistent with this, our MM/PBSA calculations indicated that Y89 contributed unfavorably to the free energy of the C6-oriented binding conformation (Figure B), further confirming its key role in substrate conformational control and site-selective reactivity. We hypothesized that reducing the hydrogen-bonding capability and steric hindrance at this position would facilitate substrate flipping and promote C6 regioselectivity. Accordingly, we introduced a Y89F mutation on the A248S/H176Y background to generate a triple mutant, aiming to bring the substrate C6 closer to nitrogen atom. The experimental results showed that the catalytic efficiency of this triple mutant was comparable to that of the A248S/H176Y double mutant. However, no significant increase in the C6 product proportion was observed, and its regioselectivity shifted markedly toward C4 (Figure D,E), with 93% selectivity. These findings indicate that further mutagenesis on the A248S/H176Y platform is considerably limited in achieving high C6 selectivity and underscore the intrinsic challenge of efficiently achieving regioselective transformation at the electronically deactivated and less-reactive C6 position.

Subsequently, we reanalyzed the A248S/H176F double mutant, which only produces 5-nitrotryptophan. This mutant may lack sufficient steric repulsion within the active pocket to drive substrate flipping, thereby limiting C6-oriented binding. To test this hypothesis, we calculated the pocket volumes of A248S/H176W, A248S/H176Y, and A248S/H176F using CASTpFold platform. The results showed that A248S/H176F has a significantly larger pocket volume of 351.54 ų relative to the other two variants (Figure F), consistent with our assumption. Although this mutant does not yet achieve effective C6 orientation, its larger pocket may provide structural optimization effects. Based on this rationale, we selected A248S/H176F as a new design starting point and, guided by previous MMPBSA energy decomposition results, introduced a Y89F mutation to expose the substrate C6 toward the electrophilic attack site of •NO₂ The resulting A248S/H176F/Y89F triple mutant successfully generated C6-nitrated products. Although the regioselectivity ability was modest (26%), this outcome validated our conformational control hypothesis and indicated room for further improvement. Taken together, our results suggest that mutagenesis strategies starting with A248S alone are insufficient for achieving high C6 selectivity. In contrast, the design based on A248S/H176F combined with Y89F substantially increased the proportion of C6 products, exceeding that of the A248S/H176Y/Y89F variant by over 20% (Figure D). Therefore, we concluded that the H176F-centered conformation provided a greater potential for achieving high regioselectivity at the C6 position of tryptophan.

2.4. Mechanism of H176F/A248F-Catalyzed C6 Tryptophan Nitration

In our previous mutagenesis screenings (Table , Figure C, A248, M88Y), and Y89F were identified as key residues influencing substrate conformation and regioselectivity. Then, we introduced M88Y and Y89F mutations into the H176F background to further enhance C6 product formation. However, the H176F/M88Y mutant completely lost catalytic activity, while the H176F/Y89F variant still exhibited detectable catalytic activity (Figure A), but no longer produced C6-nitrated products. These results indicated that in the H176F background, A248 was critical for inducing the substrate conformational shifts necessary for C6 nitration. Therefore, we performed saturation mutagenesis at A248 in the H176F background. Interestingly, both the H176F/A248F and H176F/A248T mutants achieved excellent regioselectivity, with C6 product proportions exceeding 99% (Figure A), indicating the first instance of highly selective single-step C6 nitration. Among these, H176F/A248F not only displayed the highest selectivity but also the highest total turnover number (TTN = 13.9) of all variants, demonstrating both excellent catalytic efficiency and potential for industrial applications. Moreover, we further explored additional mutations on top of H176F/A248F to generate triple mutants, however, all such variants exhibited significantly reduced activity or no detectable turnover. Therefore, H176F/A248F represents the optimal mutant identified in this study.

4.

Catalytic performance and C6 selectivity of H176F/A248F. (A) Catalytic activity of H176F-based mutants. (B) Binding free energies of C6-oriented tryptophan in WT vs H176F/A248F from MD. (C) Active-site interactions in MD-derived ferric peroxynitrite tryptophan complex of H176F (PDB: 5D3U). (D) Active-site interactions in MD-derived ferric peroxynitrite tryptophan complex of H176F/A248F crystal structure. Dashed lines indicate hydrophobic interactions; solid lines, hydrogen bonds. (E) QM/MM energy profiles for WT and H176F/A248F catalysis.

To elucidate the precise recognition mechanism underlying C6 nitration by the H176F/A248F mutant, we characterized the mutant–substrate complex using X-ray crystallography and successfully resolved a high-resolution structure at 1.53 Å (PDB ID: 9UYC). The related crystallographic statistics are summarized in Table S3. Based on this structure, we further analyzed the substrate-binding mode and key interactions using molecular docking and 100 ns MD simulations. MM/PBSA calculations indicated that the binding free energy of tryptophan to this mutant was significantly lower than that of the wild-type (Figure B), demonstrating enhanced substrate binding and confirming the critical role of the active-pocket environment in controlling regioselectivity.

To understand how the pocket environment governs site selectivity, we performed a systematic comparison of the substrate-binding modes of H176F and H176F/A248F. Arnold et al. showed that H176F alone directs nitration to C5. In the H176F single mutant, the C7 position of the substrate on the phenyl ring engaged in hydrophobic interactions with F395 and F176, whereas the C5 position engaged in hydrophobic interactions with A245 (Figure C). Additionally, the amino and carboxyl groups of the substrate formed hydrogen bonds and salt bridges with R59, respectively. Collectively, these interactions stabilized the C5 position of the substrate nearest to the N atom of peroxynitrite (Figure C). Mutation of A248 to the bulkier F248 increases steric crowding in the pocket, which in turn induces a conformational shift of F395. This rearrangement redirects its hydrophobic interaction from the substrate’s C7 region toward the C6 position. This change induces an approximately 90° rotation of the substrate, which is further promoted by steric repulsion from F248, displacing it away from A245 and toward Y89. A new hydrogen bond was formed between Y89 and the indole nitrogen of the substrate (Figure D), collectively positioning the phenyl ring C6 adjacent to the N atom of peroxynitrite. Hydrogen-bond interactions between the amino and carboxyl groups of the substrate and R59 partially stabilized the C6–N distance, thereby enabling highly selective nitration of the C6 position of tryptophan.

Finally, to elucidate the catalytic mechanism underlying C6-selective nitration, QM/MM reaction models were constructed for both the wild-type TxtE and the H176F/A248F mutant based on equilibrated Trp-bound structures. The ^•NO₂ radical was then introduced as the initial reactive species to directly explore its escape pathway and subsequent reactivity. QM/MM calculations revealed that, in the wild-type TxtE, the transition-state energy barrier for C6 nitration is as high as 54.3 kcal·mol^–1 (Figure E), far exceeding the feasible range for typical biocatalytic reactions. This finding is consistent with the MMPBSA results mentioned above, which indicated that C6 binding is thermodynamically disfavored and that C6 nitration in the WT background is highly unfavorable, both kinetically and thermodynamically. In contrast, the H176F/A248F double mutant dramatically lowers the barrier to 17.9 kcal·mol^–1, enabling highly selective C6 nitration. This demonstrates that the precise modulation of the active-pocket geometry and substrate interactions through mutagenesis can induce substrate conformational rearrangement, thereby altering regioselectivity. Further analysis of the transition-state structures showed that the distance between the reactive ^•NO₂ and the C6 atom was shortened to 2.1 Å in H176F/A248F, vs 2.3 Å in the WT. This indicates that the double mutation reshaped the active pocket conformation to improve substrate positioning, effectively promoting highly selective C6 nitration. These results demonstrate that our regioselectivity-guided design platform successfully enables the highly selective C6 nitration of tryptophan, validating that precise modulation of the active-pocket environment can drive targeted site-specific functionalization. This achievement not only overcomes the long-standing challenge associated with C6 nitration but also provides a feasible strategy for the future development of selective C7 nitration of L-tryptophan.

3. Conclusion

In summary, this study employed a computer-aided regioselective design strategy to functionally remodel P450 TxtE, yielding the H176F/A248F double mutant that, for the first time, has overcame the long-standing challenge of achieving C6 nitration on the indole ring of L-tryptophan. Structural analysis combined with QM/MM calculations revealed that steric hindrance–driven active-site reorganization and substrate conformational flipping are the key mechanisms enabling the high selectivity at the C6 position. Moreover, integrating our findings with previous studies on TxtE catalysis, we discovered that precise modulation of key active-site residues can “turn the clock hands” to reshape the substrate-binding conformation, thereby enabling controllable migration of the nitration site between C4, C5, and C6. Based on these insights, we propose the ‘clock-turning’ catalytic regulation of TxtE to achieve precise regioselectivity. This work not only endows TxtE with unprecedented catalytic regioselectivity but also validates the feasibility of rationally reprogramming enzyme-catalyzed regioselectivity. Importantly, the proposed model and established design platform provide a new conceptual framework and a generalizable strategy for site-selective functionalization of aromatic compounds, with promising potential for the selective synthesis of pharmaceuticals and fine chemicals.

4. Materials and Methods

4.1. Construction and Mutation of the TxtE-BM3R Plasmid

This study obtained the gene sequences of P450 TxtE (GenBank NO.: CBG70284.1) and P450 BM3 , (GenBank NO.: J04832.1) from the GenBank database. Nucleotide sequence analysis of P450 TxtE BM3R was performed using Ape software. The expression vector used for P450 TxtE BM3R was pET28a, with Nco I and Xho I as restriction sites. A Sac I restriction site was introduced between TxtE and BM3R to facilitate the construction of subsequent mutant strains. A 6× His tag was introduced at the N-terminus via primer design. Since TxtE BM3R consists of TxtE and the reductase domain of P450 BM3, a fusion PCR approach was used to construct the recombinant plasmid of P450 TxtE BM3R.TxtE-pET28a and BM3-pET28a were synthesized and used as templates for PCR amplification using a PrimerStar Premix polymerase. The PCR products obtained from the above reactions were subjected to agarose gel electrophoresis. DNA bands corresponding to the expected sizes of the target genes were excised and purified using the PCR/Gel Purification Kit from FAVORGEN. The upstream and downstream fragments obtained from gel extraction were fused using fusion PCR. The resulting PCR product was subjected to agarose gel electrophoresis, and DNA bands corresponding to the expected size of the target gene were excised and purified. The purified target gene and vector were then digested using a double-enzyme digestion system. After incubation at 37 °C for 1 h, the digestion products were separated by agarose gel electrophoresis, and bands of the correct sizes for the target gene and vector were recovered. The target gene was then ligated into the vector using T4 DNA ligase. The ligation product containing the target gene was transformed into competent cells using the heat shock method.

4.2. Expression and Purification of TxtE-BM3R

TxtE-BM3R was expressed using Escherichia coli BL21 (DE3) as the host strain. The cells were cultured in LB medium containing 50 μg/mL kanamycin sulfate. A single colony was picked and inoculated into 10 mL of LB medium, followed by incubation at 37 °C with shaking at 220 rpm for 12 h. The culture was then transferred into 400 mL of fresh medium and grown until the optical density at 600 nm (OD₆₀₀) reached 0.6–0.8. Induction was carried out by adding 0.1 mM IPTG and 0.2 mM 5-ALA, followed by incubation at 20 °C. The bacterial cells were harvested by centrifugation at 8000g for 5 min. During the protein purification process, all samples were kept on ice. The harvested cells were resuspended in Buffer A (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 30 mM imidazole, 3 mM β-mercaptoethanol, and 10% glycerol) and lysed by ultrasonication. The lysate was centrifuged at 18,000g for 40 min, and the supernatant was filtered through a 0.22 μm membrane. Buffer A and Buffer B (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 300 mM imidazole, 3 mM β-mercaptoethanol, and 10% glycerol) were linearly mixed and used for elution on the AKTA protein purification system to obtain high-purity protein. The eluted protein was then buffer-exchanged into an imidazole-free solution using a 30 kDa MWCO centrifugal filter device. Protein bands were verified by SDS-PAGE.

4.3. Synthesis of Products and Detection

4-Nitro-dl-tryptophan and 5-nitro-dl-tryptophan were purchased from Sigma-Aldrich. 6-nitro-dl-tryptophan was synthesized using the nitrotryptophan synthase Pf0A9 E104G as the catalyst. Further analysis was performed using 600 MHz ¹H NMR spectroscopy­(the results are in SI).The Waters e2695 high performance liquid chromatography (HPLC) system was used to detect nitrated products. To achieve complete separation of all products, a phenyl-hexyl column (250 mm × 4.6 mm, 5 μm, Phenomenex) was employed. Acetonitrile and water were used as the mobile phase, and detection was performed using a PDA detector with full-wavelength scanning. 4-nitro-dl-tryptophan exhibits a characteristic absorption peak at 400 nm, while 5-nitro-dl-tryptophan shows a distinct peak at 330 nm, consistent with previous studies. , In contrast, 6-nitro-dl-tryptophan displays a broad and prominent peak at 334 nm, which is significantly different from the other nitrated products.

4.4. Purified Enzyme and Control Reactions

The reaction mixture (total volume 300 μL) contained 10 μM purified enzyme, 1 mM l-tryptophan, 2 mM NOC-5, 2 mM NADP⁺, 10 mM glucose, and 25 U glucose dehydrogenase (GDH), with buffer added to the final volume. Reactions were incubated at 20 °C and shaken at 200 rpm for 12 h. The product detected by HPLC.

4.5. Crystallization and Data Collection

The enzyme mixture and the substrate l-tryptophan were combined to a final enzyme concentration of 15 mg/mL and an l-tryptophan concentration of 10 mM, followed by incubation on ice for 30 min. Crystallization screening was performed using the Screens crystallization kit (HR2–144), which includes 96 different buffer conditions. For each condition, 75 μL of reservoir solution was added to the well of a 96-well sitting-drop plate. Subsequently, 0.8 μL of the enzyme–substrate mixture was mixed with 0.8 μL of the corresponding reservoir solution in each well. The plate was sealed tightly with adhesive film and incubated at 20 °C in a constant-temperature incubator. After 1 week, crystal growth was examined under a microscope (0.2 M sodium potassium tartrate tetrahydrate and 20% w/v poly­(ethylene glycol) 3350). Crystals were harvested and transferred into a cryoprotectant solution containing 50% glycerol, then rapidly flash-frozen in liquid nitrogen. X-ray diffraction data were collected at beamline BL02U1 of the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) and processed using the XDS program. Model building and refinement were carried out using Refmac5 and Coot. ,

4.6. Classical MD Simulation

The initial structure of H176FA248F and WT were derived from its crystal structure (PDB code: 9UYC and 4TPO). The AMBER14SB , force field was used to generate parameters for the protein, and a TIP3P tank with a 12 Å buffer was chosen as the solvent environment. The force field for the Fe­(IV)–O state was parametrized using the “MCPB.py”tool from AmberTools18. Sodium ions were added to maintain a neutral pH. The most rapid descent and conjugate gradient methods were applied sequentially to optimize the solvent and solute structures and minimize the energy. Subsequently, the entire system was heated to 300 K under the constraint of 10 kcal/(mol Ų). The SHAKE algorithm was used to constrain chemical bonds involving hydrogen atoms. The heating stage lasted 100 ps (0.1 ns), with a time step of 2 fs, gradually bringing the system to the desired temperature. During this stage, the temperature was controlled using the Langevin dynamics algorithm, with a damping coefficient to stabilize the system’s temperature. Pressure was maintained using the Berendsen barostat with a pressure relaxation time constant. Finally, a 100 ns simulation was performed without any constraint in the NPT synthesis

4.7. QM Model Calculations

Density functional theory (DFT) calculations for the QM model were performed using Gaussian 16 software. Geometry optimization and frequency calculations were conducted at the B3LYP/def2-SVP level, with solvent effects of water accounted for using the SMD continuum solvation model. After structure optimization, more accurate single-point energies were computed using the larger def2-TZVP basis set at the B3LYP level. All QM calculations included dispersion corrections calculated using Grimme’s D3 method.

4.8. QM/MM Methodology

All QM/MM calculations were performed using ChemShell, combining ORCA as the QM code and DL_POLY as the MM code. Residues within 7.0 Å of the Fe atom were included in the active region to allow relaxation during geometry optimization, which include both QM atoms (about 30 atoms) and MM atoms (about 900 atoms). The QM region contains the OON and substrate. The electronic embedding scheme was employed to include the polarizing effect of the enzyme environment on the QM region. For the QM region, the B3LYP functional was utilized, B3LYP has been proven as a successful functional for studying Fe (IV)/2OG-dependent enzymes. − For geometry optimization and frequency calculations, the all-electron basis set Def2SVP was used. The energies were further corrected with the larger basis set Def2TZVP. The DL-FIND optimizer was used in the geometry optimization.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01378.

• Detailed information on product characterization, catalytic performance evaluation, and computational analysis, including HPLC/UPLC-MS, NMR, HRMS, standard curves, MD simulations, and QM/MM calculations (PDF)

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X.Y., Y.P., and T.M. contributed equally to this work. X.Y., Y.P., T.M. carried out the experiments under the supervision of L.Z., B.G. X.Y. and T.M. resolved the crystal structures under the supervision of L.Z., B.G. Z.Y. and Y.C. performed the computational studies. X.Y. and T.M.wrote the manuscript, and all authors discussed the results and commented on the manuscript.

The authors declare no competing financial interest.