Saturation Mutagenesis Research Articles

Promiscuity Guided Evolution of Decarboxylative Aldolases for Synthesis of Tertiary γ-Hydroxy Amino Acids.

Many applications of enzymes benefit from activity on structurally diverse substrates. Here, we sought to engineer the decarboxylative aldolase UstD to perform a challenging C-C bond forming reaction with ketone electrophiles. The parent enzyme had only low levels of activity, portending multiple rounds of directed evolution and a possibility that mutations may inadvertently increase the specificity of the enzyme for a single model screening substrate. We show how to intentionally guide UstD towards generality through multi-generational directed evolution using substrate-multiplexed screening (SUMS). Mutations outside of the active site that impact catalytic function were immediately revealed by shifts in promiscuity, even when the overall activity was lower. By re-targeting these distal residues that couple to the active site with saturation mutagenesis, broadly activating mutations were readily identified. When analyzing active site mutants, SUMS identified both specialist enzymes that would have more limited utility as well as generalist enzymes with complementary activity on diverse substrates. These new UstD enzymes catalyze convergent synthesis of non-canonical amino acids bearing tertiary alcohol side chains. This methodology is easy to implement and enables the rapid and effective evolution of enzymes to catalyze desirable new functions.

Engineering of Cyclodextrin Glucosyltransferase from Paenibacillus macerans for Improved Regioselectivity and Product Specificity Toward Hydroxyflavone Glycosylation

The regioselective glycosylation and product specificity of hydroxyflavonoid compounds profoundly influences their biological activity and stability, offering significant therapeutic potential. However, most cyclodextrin glucosyltransferases (CGTases) inherently lack regioselectivity and product specificity for flavone glycosylation. Herein, a CGTase from Paenibacillus macerans was engineered for enhanced glycosylation regioselectivity and product specificity by combining molecular docking analysis and saturation mutagenesis strategies. K232L (favoring 4′-and 6-hydroxyflavones) and K232V (favoring 7-hydroxyflavone) were identified with distinct preferences. In addition, H233Y (preferring for 4′-hydroxyflavones), H233T (preferring for 6′-hydroxyflavones), and H233K (preferring for 7′-hydroxyflavones) also demonstrated distinct regioselectivity. These variants further exhibited enhanced hydrolytic activity, enabling the efficient production of short sugar-chain glycosides. Molecular dynamics (MDs) simulations revealed that the variants adopted optimized catalytic conformations with increased loop region flexibility near the binding pocket, enhancing substrate accessibility. These findings underscore the pivotal roles of K232 and H233 in broadening the substrate scope of CGTase and offer valuable guidance for enzyme engineering targeting regioselective glycosylation.

Enantiodivergent Synthesis of Both (R)- and (S)-Heteroaryl Aldols by Rationally Engineered Aldolases.

Aldolases, especially 2-deoxyribose-5-phosphate aldolase (DERA) enzymes, have been widely employed to access key chiral precursors for various active pharmaceutical ingredients (APIs). This has been enabled by expanding their substrate scope toward non-natural acceptors and donors via protein engineering. In this study, we endeavored to broaden the acceptor substrate scope of DERA from Geobacillus sp. (DERAGeo) toward the heteroaryl aldehydes through a rational protein engineering approach. We successfully performed iterative saturation mutagenesis of DERAGeo, resulting in two enantiocomplementary variants, viz., (R)-selective DERAGeo-S185G and (S)-selective DERAGeo-T12I/S185A, with enhanced catalytic efficiencies and enantioselectivities. Remarkably, the natural enantioselectivity of DERAGeo was reversed by a single mutation (S185G). The synthetic applicability of the mutants was demonstrated by conducting aldol reactions on a semipreparative scale, from which both (R)- and (S)-enantiomers of heteroaryl aldols were isolated with high yields (up to 99%) and excellent enantiopurities (up to 99:1 e.r.).

Computational mining of a novel Acetyl‐CoA synthetase for the biosynthesis of unnatural Phenylalanine butyramide

Phenylalanine butyramide (FBA) is an unnatural amide with the potential to be a postbiotic derivative due to its improved organoleptic and physicochemical properties compared to butyric acid and salts. However, a green biosynthesis method to prepare FBA has not been studied to date. In this study, we mined a novel Acetyl‐CoA synthetase from Methanocella arvoryzae (MethACS) for the synthesis of FBA using a sequence and structure‐guided database mining approach that combines solubility prediction with binding free energy of the protein. MethACS exhibits excellent catalytic performance, pH stability, and good thermophilicity. 0.35 g/L FBA could be produced by MethACS with a 100% conversion rate when the concentration ratio of butyric acid to phenylalaninamide was >1:4. Through density‐functional theory calculations and molecular dynamics simulation, we gained insight into the mechanism of the synthesis of FBA catalyzed by MethACS. Trp264 was further identified as a crucial residue influencing the substrate specificity by saturation mutagenesis. Substrate profiling revealed that MethACS can catalyze the formation of amide derivatives of short‐ and medium‐chain fatty acids. This study offers an efficient approach for investigating the biosynthesis of unnatural substances.

Site-saturation mutagenesis of 500 human protein domains

Missense variants that change the amino acid sequences of proteins cause one-third of human genetic diseases1. Tens of millions of missense variants exist in the current human population, and the vast majority of these have unknown functional consequences. Here we present a large-scale experimental analysis of human missense variants across many different proteins. Using DNA synthesis and cellular selection experiments we quantify the effect of more than 500,000 variants on the abundance of more than 500 human protein domains. This dataset reveals that 60% of pathogenic missense variants reduce protein stability. The contribution of stability to protein fitness varies across proteins and diseases and is particularly important in recessive disorders. We combine stability measurements with protein language models to annotate functional sites across proteins. Mutational effects on stability are largely conserved in homologous domains, enabling accurate stability prediction across entire protein families using energy models. Our data demonstrate the feasibility of assaying human protein variants at scale and provides a large consistent reference dataset for clinical variant interpretation and training and benchmarking of computational methods.

Improving the binding affinity of plastic degrading cutinase with polyethylene terephthalate (PET) and polyurethane (PU); an in-silico study.

Plastic pollution is a worrying problem, and its degradation is a laborious process. Although enzymatic plastic breakdown is a sustainable method, drawbacks such as numerous plastic kinds of waste make the degradation challenging. Therefore, a multi-plastic degrading (MPD) enzyme becomes necessary. In this in-silico study, microorganisms and their enzymes that are known to degrade plastic polymers such as PET, PU, PVC, and PE were identified to assess their MPD capability. The cutinase of Thermobifida fusca was found to degrade both PET and PU polymers. The crystallized structure of cutinase was retrieved from PDB, and PET, PU ligands were docked using Schrodinger. However, the interactions between cutinase and the ligands were not efficient, as evidenced by the docking scores of -4.047 and -4.993 for PET and PU, respectively. Nevertheless, the interaction of the cutinase's active site with the ligands by hydrogen bond formation was promising. In this work, unconserved regions of cutinase were identified as potential mutation sites to enhance binding efficiency. In-silico Alanine Scanning Mutagenesis (ASM) and Site Saturation Mutagenesis (SSM) were performed as screening tests to find variants of cutinase with better docking scores for both ligands, specifically S136D, N28M, and S136Q. Molecular Dynamic Simulation (MDS) was performed for Wild Type (WT) cutinase, variants, and their respective complexes formed with the ligands. This simulation indicated the compactness, stability, and minimal energy of the variant complexes compared to WT complexes. Subsequent in vitro studies can ensure the improved degradation of both PET and PU by the variants.

Advancing PET-Degrading Enzymes through Directed Evolution to Combat Plastic Pollution

Plastic pollution poses a significant environmental challenge due to its persistence and widespread distribution. Among various types of plastic, polyethylene terephthalate (PET) is especially problematic due to its resistance to natural degradation. PET-degrading enzymes, particularly PETases and cutinases, have emerged as promising solutions for enzymatic plastic recycling. However, their native catalytic efficiency and thermostability are limited. Directed evolution has enabled the development of improved enzyme variants through techniques such as error-prone PCR, DNA shuffling, and saturation mutagenesis. This review highlights recent advances in engineering Cutinase and PETases, focusing on enhancing catalytic efficiency and thermostability for PET plastic degradation by directed evolution. Key engineered variants, including HotPETase and optimized leaf-branch compost cutinase (LCC) mutants, demonstrate significant progress toward sustainable plastic recycling through enzymatic means.

Production of 2-O-α-d-glucopyranosyl-l-ascorbic acid using sucrose phosphorylase by semi-rational design

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is often substituted for l-ascorbic acid (L-AA) in health- and skincare products due to its enhanced stability and comparable antioxidant. Enzymatic catalysis for AA-2G is gaining widespread interest and sucrose phosphorylases (SPase) have shown promise in achieving higher yields. To enhance AA-2G synthesis, we screened and identified the SPase from Bifidobacterium longum (BlSPase) as the starting enzyme, with an optimal pH of 5.4 and temperature of 45 °C for AA-2G synthesis. Subsequently, we conducted semi-rational design on BlSPase based on modeling and molecular docking. L-AA was docked with the BlSPase model to analyze key residues in the ‘loop-door’ domain and substrate-binding pocket. Through alanine scanning and saturation mutagenesis, a mutant library was created. After single-point and composite mutation, L341V/V346P was identified as the optimal variant. Ultimately, within 72 h, we achieved yield of 358.6 g/L AA-2G with a molar conversion of L-AA reaching 75.7 %.

Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning.

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.

Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection.

Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of ThAdo, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.

Computational Mutagenesis of GPx7 and GPx8: Structural and Stability Insights into Rare Genetic and Somatic Missense Mutations and Their Implications for Cancer Development.

Background/Objectives: Somatic and genetic mutations in glutathione peroxidases (GPxs), including GPx7 and GPx8, have been linked to intellectual disability, microcephaly, and various tumors. GPx7 and GPx8 evolved the latest among the GPx enzymes and are present in the endoplasmic reticulum. Although lacking a glutathione binding domain, GPx7 and GPx8 possess peroxidase activity that helps the body respond to cellular stress. However, the protein mutations in these peroxidases remain relatively understudied. Methods: By elucidating the structural and stability consequences of missense mutations, this study aims to provide insights into the pathogenic mechanisms involved in different cancers, thereby aiding clinical diagnosis, treatment strategies, and the development of targeted therapies. We performed saturated computational mutagenesis to analyze 2926 and 3971 missense mutations of GPx7 and GPx8, respectively. Results: The results indicate that G153H and G153F in GPx7 are highly destabilizing, while E93M and W142F are stabilizing. In GPx8, N74W and G173W caused the most instability while S70I and S119P increased stability. Our analysis shows that highly destabilizing somatic and genetic mutations are more likely pathogenic compared to stabilizing mutations. Conclusions: This comprehensive analysis of missense mutations in GPx7 and GPx8 provides critical insights into their impact on protein structure and stability, contributing to a deeper understanding of the roles of somatic mutations in cancer development and progression. These findings can inform more precise clinical diagnostics and targeted treatment approaches for cancers.

Engineering a Binding Peptide for Oriented Immobilization and Efficient Bioelectrocatalytic Oxygen Reduction of Multicopper Oxidases.

Enzymatic fuel cells (EFCs) are emerging as promising technologies in renewable energy and biomedical applications, utilizing enzyme catalysts to convert the chemical energy of renewable biomass into electrical energy, known for their high energy conversion efficiency and excellent biocompatibility. Currently, EFCs face challenges of poor stability and catalytic efficiency at the cathodes, necessitating solutions to enhance the oriented immobilization of multicopper oxidases for improved heterogeneous electron transfer efficiency. This study successfully identified a surface-binding peptide (SBP, 13 amino acids) derived from a methionine-rich fragment (MetRich, 53 amino acids) in E. coli CueO through semirational design. The first phase of engineering focused on the structural characteristics of MetRich, pinpointing fragment N394-H406 (SBP 1.0, corresponding to variant CueO-M12) as the key region dominating the binding. Subsequent site-saturation mutagenesis, combined with electrochemical screening, yielded three variants, and among them, the variant CueO-M12-1 (CueO-M12 H398I) exhibited a more uniform favorable orientation with a 1.38-fold increase in current density. Further electrocatalytic kinetics analysis revealed a significant 21.2-fold improvement in kinetics current density (Jk) compared with that of CueO-WT, leading to the development of SBP 2.0. When SBPs were fused to laccase from Bacillus pumilus (BpL) and fungal bilirubin oxidase from Myrothecium verrucaria (MvBOD), respectively, they transformed a sluggish adsorption process into a rapid and oriented one. In addition, compared with SBP 1.0, SBP 2.0 endows BpL and MvBOD with enhanced electrocatalytic capabilities for oxygen reduction and glucose/O2 EFC performance. The engineered SBPs are promising for serving as a versatile "glue" to enable the immobilization of oxidoreductases in an oriented manner, which leads to a breakthrough in bioelectrocatalysis and thereby overcoming the current bottleneck of EFCs.

Targeted Directed Evolution of an α-L-Rhamnosidase on Hesperidin Through Six-Codon Combinatorial Mutagenesis

Targeted saturation mutagenesis at the residues located at the substrate-binding pocket for generating focused libraries has emerged as the technique of choice for enzyme engineering, but choosing the optimal residue number of the randomization site and the reduced amino acid alphabet to minimize the labor-determining screening effort remains a challenge. Herein, we propose the six-codon combinatorial mutagenesis (SCCM) strategy by using the BMT degeneracy codons encoding six amino acids with different chemical properties as the building blocks for the randomization of the amnio acid motif. SCCM requires only a small library of 646 clones for 95% coverage at the three-residue motif compared to conventional NNK degeneracy codons encoding all 20 canonical amino acids and requiring the screening of nearly 100,000 clones. SCCM generates a suitable number of mutant libraries, providing a new strategy for reducing the screening workload of saturated combination mutations in enzyme engineering. Using this approach, the α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron had been successfully engineered for improving the hydrolytic activity on natural flavonoid diglycoside hesperidin via targeted directed evolution at the motifs positioning the entrance of the substrate-binding pocket. The results indicate that the conversion rates of the four mutants on hesperidin were increased by more than 30% compared with the wild type using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of the mutant TM1-6-F5 was 1.4-fold higher than that of the wild type.

Artificial Gold Enzymes Using a Genetically Encoded Thiophenol‐Based Noble‐Metal‐Binding Ligand

AbstractIncorporating noble metals in artificial metalloenzymes (ArMs) is challenging due to the lack of suitable soft coordinating ligands among natural amino acids. We present a new class of ArMs featuring a genetically encoded noble‐metal‐binding site based on a non‐canonical thiophenol‐based amino acid, 4‐mercaptophenylalanine (pSHF), incorporated in the transcriptional regulator LmrR through stop codon suppression. We demonstrate that pSHF is an excellent ligand for noble metals in their low oxidation states. The corresponding gold(I) enzyme was characterised by mass spectrometry, UV/Vis spectroscopy and X‐ray crystallography and successfully catalysed hydroamination reactions of 2‐ethynyl anilines with turnover numbers over 50. Interestingly, two equivalents of gold(I) per protein dimer proved to be required for activity. Up to 98 % regioselectivity in the hydroamination of an ethynylphenylurea substrate was observed, yielding the corresponding phenyl‐dihydroquinazolinone product, consistent with a π‐activation mechanism by single gold centres. The ArM was optimized by site saturation mutagenesis using an on‐bead screening protocol. This resulted in a single mutant that showed higher activity for one class of substrates. This work brings the power of noble‐metal catalysis into the realm of enzyme engineering and establishes thiophenols as alternative ligands for noble metals, providing new opportunities in coordination chemistry and catalysis.

Active Site Engineering of a Glycerol Dehydrogenase Improves its Oxidative Activity and Scope Toward Glycerol Derivatives.

Regioselective oxidation of glyceryl alkyl ethers is of utmost importance for the fabrication of substituted hydroxy ketones and enantiopure 1,2-diols as green solvents and pharmaceutical building blocks, respectively. An engineered glycerol dehydrogenase from Bacillus stearothermophilus was described to perform the regioselective oxidation of alkyl glycerol ethers, identifying position 252 as key for accepting larger substrates than glycerol. In this work, we further engineer that position through partial saturation mutagenesis to broaden the substrate scope toward other glycerol derivatives, improving enzyme kinetics and minimizing product inhibition. In particular, the BsGlyDH-L252S variant becomes the most efficient biocatalyst for the deracemization of alkyl glyceryl ethers in a two-step, one-pot immobilized system. The discovery and use of these alternative mutants of GlyDH opens the road to more applications and increases the enzymatic toolbox for the modifications of glyceryl ethers.

Engineering FRET biosensor for H3K9 acetylation imaging in single living cells

Histone acetylation is an important epigenetic modification that governs gene expression, chromatin changes in stress response, and cell fate transition. FRET biosensors have been developed for various epigenetic events to enable spatiotemporal tracking of sub-cellular signaling events. Previously reported histone H3 acetylation biosensor recognizing two acetyl residues lacked specificity. In this study, using a single bromodomain of the BRD4, we have developed a genetically encoded H3K9ac biosensor. We systematically investigated different combinations of the BET family protein as binding domains and performed site-saturated mutagenesis to optimize the biosensor, achieving a dynamic FRET change up to 30% under TSA treatment. With the application of the optimized H3K9ac biosensor, we revealed different basal active chromatin architectures in invasive tumor cells compared to benign tumor cells. Furthermore, we found that H3K9ac level increased dramatically when cancer cells passed through microchannels, which models the physical constraints and mechanical microenvironmental conditions that cancer cells encounter when passing through narrow spaces within the body. This result highlights the chromatin plasticity in response to external mechanical stresses. In summary, our H3K9ac biosensor provides a versatile tool for mechanistic investigation of cell fate transition in cancer and mechanotransduction.Graphical

A recombinant L-threonine aldolase with high catalytic efficiency for the asymmetric synthesis of L-threo-phenylserine and L-threo-4-fluorophenylserine.

To develop robust variants of L-threonine aldolases (L-TAs), potent catalysts for synthesizing asymmetric β-hydroxy-α-amino acids, it is necessary to identify critical residues beyond the known active site residues. Through virtual screening, a neglected residue Asn305, was identified as critical for catalytic efficiency. Subsequent site-saturation mutagenesis led to a potent variant N305R which exhibited excellent conversions of 88%conv (87%de) and 80%conv (94%de) for the synthesis of L-threo-phenylserine and L-threo-4-fluorophenylserine respectively. This variant not only outperformed the template enzyme, but also represented a promising L-TA for synthesizing the two β-hydroxy-α-amino acids. It was suggested that Arg305 of the variant N305R generated strong cation-arene interaction and electrostatic force with the intermediates, leading to strengthened binding, enhanced L-threo favored orientation and wider entrance. Our work not only provided an excellent variant N305R, but also suggested the crucial function of a neglected residue Asn305, which offered valuable experiences for other L-TA studies.

Engineering the Reaction Pathway of a Non-heme Iron Oxygenase Using Ancestral Sequence Reconstruction.

Non-heme iron (FeII), α-ketoglutarate (α-KG)-dependent oxygenases are a family of enzymes that catalyze an array of transformations that cascade forward after the formation of radical intermediates. Achieving control over the reaction pathway is highly valuable and a necessary step toward broadening the applications of these biocatalysts. Numerous approaches have been used to engineer the reaction pathway of FeII/α-KG-dependent enzymes, including site-directed mutagenesis, DNA shuffling, and site-saturation mutagenesis, among others. Herein, we showcase a novel ancestral sequence reconstruction (ASR)-guided strategy in which evolutionary information is used to pinpoint the residues critical for controlling different reaction pathways. Following this, a combinatorial site-directed mutagenesis approach was used to quickly evaluate the importance of each residue. These results were validated using a DNA shuffling strategy and through quantum mechanical/molecular mechanical (QM/MM) simulations. Using this approach, we identified a set of active site residues together with a key hydrogen bond between the substrate and an active site residue, which are crucial for dictating the dominant reaction pathway. Ultimately, we successfully converted both extant and ancestral enzymes that perform benzylic hydroxylation into variants that can catalyze an oxidative ring-expansion reaction, showcasing the potential of utilizing ASR to accelerate the reaction pathway engineering within enzyme families that share common structural and mechanistic features.

All-Atom Perspective of the DENV-3 Methyltransferase Inhibition Mechanism.

The Dengue virus (DENV) is an enveloped, single-stranded RNA virus with several antigenically distinct serotypes (DENV-1 to DENV-5). Dengue fever, as a major public health threat transmitted by mosquitoes, affects millions of people worldwide (especially in tropical and subtropical regions). Toward drug developments of DENV, the nonstructural protein 5 methyltransferase (MTase) serves as an attractive target. The MTase transforms S-adenosyl methionine to S-adenosyl homocysteine (SAH), which is thereby selected as the target with which external drugs compete with. In this work, using alanine scanning with generalized Born and interaction entropy (ASGB-IE), we provide an all-atom perspective of the protein-ligand interactions formed by DENV-3 MTase and SAH derivatives. Residues with consistently high contributions to stabilization are summarized, and the general DENV-3 MTase inhibition mechanism is elucidated. Additionally, the mutational impact on binding thermodynamics is found to be entropy-driven. We also highlight the advantage of the ASGB-IE method for affinity estimation compared to standard end-point protocols, which is highly related to the selection of interfacial residues in free energy estimation. Finally, we performed a thorough scan of the mutational space on critical sites (saturation mutagenesis) and identified 14 mutants causing resistance to the current inhibitors.

Mechanistic insights into allosteric regulation of the reductase component of p-hydroxyphenylacetate 3-hydroxylase by p-hydroxyphenylacetate: a model for effector-controlled activity of redox enzymes.

Understanding how an enzyme regulates its function through substrate or allosteric regulation is crucial for controlling metabolic pathways. Some flavin-dependent monooxygenases (FDMOs) have evolved an allosteric mechanism to produce reduced flavin while minimizing the use of NADH and the production of harmful hydrogen peroxide (H2O2). In this work, we investigated in-depth mechanisms of how the reductase component (C1) of p-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from Acinetobacter baumanii is allosterically controlled by the binding of HPA, which is a substrate of its monooxygenase counterpart (C2). The C1 structure can be divided into three regions: the N-terminal domain (flavin reductase); a flexible loop; and the C-terminal domain, which is homologous to NadR, a repressor protein having HPA as an effector. The binding of HPA to NadR induces a conformational change in the recognition helix, causing it to disengage from the NadA gene. The HPA binding site of C1 is located at the C-terminal domain, which can be divided into five helices. Molecular dynamics simulations performed on HPA-docked C1 elucidated the allosteric mechanism. The carboxylate group of HPA maintains the salt bridge between helix 2 and the flexible loop. This maintenance shortens the loop between helices 2 and 3, causing helix 3 to disengage from the N-terminal domain. The aromatic ring of HPA induces a conformational change in helices 1 and 5, pulling helix 4, analogous to the recognition helix in NadR, away from the N-terminal domain. A Y189A mutation, obtained from site-saturation mutagenesis, confirms that HPA with an unsuitable conformation cannot induce the conformational change of C1. Additionally, an HPA-independent effect is observed, in which Arg20, an NADH binding residue on the N-terminal domain, occasionally disengages from helix 4. This model provides valuable insights into the allosteric regulation of two-component FDMOs and aromatic effector systems.