On the engineering of reductase-based-monooxygenase activity in CYP450 peroxygenases.

Recent bioengineering of CYP450OleT shows that peroxide-based CYP450OleT can be converted to a reductase-based self-sufficient enzyme, which is capable of showing efficient hydroxylation and decarboxylation activity for a wide range of substrates. The so-generated enzyme creates several mechanistic puzzles: (A) as CYP450 peroxygenases lack the conventional acid-alcohol pair, what is the source of two protons that are required to create the ultimate oxidant Cpd I? (B) Why is it only CYP450OleT that shows the reductase-based activity but no other CYP members? The present study provides a mechanistic solution to these puzzles using comprehensive MD simulations and hybrid QM/MM calculations. We show that the fusion of the reductase domain to the heme-binding domain triggers significant conformational rearrangement, which is gated by the propionate side chain, which constitutes a new water aqueduct via the carboxylate end of the substrate that ultimately participates in Cpd I formation. Importantly, such well-synchronized choreographies are controlled by remotely located Tyr359, which senses the fusion of reductase and communicates to the heme domain via non-covalent interactions. These findings provide crucial insights and a broader perspective which enables us to make a verifiable prediction: thus, the catalytic activity is not only limited to the first or second catalytic shell of an enzyme. Furthermore, it is predicted that reinstatement of tyrosine at a similar position in other members of CYP450 peroxygenases can convert these enzymes to reductase-based monooxygenases.

Amyloidogenic Propensity of Metabolites in the Uric Acid Pathway and Urea Cycle Critically Impacts the Etiology of Metabolic Disorders.

Novel insights into the etiology of metabolic disorders have recently been uncovered through the study of metabolite amyloids. In particular, inborn errors of metabolism (IEMs), including gout, Lesch-Nyhan syndrome (LNS), xanthinuria, citrullinemia, and hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, are attributed to the dysfunction of the urea cycle and uric acid pathway. In this study, we endeavored to understand and mechanistically characterize the aggregative property exhibited by the principal metabolites of the urea cycle and uric acid pathway, specifically hypoxanthine, xanthine, citrulline, and ornithine. Employing scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), we studied the aggregation profiles of the metabolites. Insights obtained through molecular dynamics (MD) simulation underscore the vital roles of π-π stacking and hydrogen bonding interactions in the self-assembly process, and thioflavin T (ThT) assays further corroborate the amyloid nature of these metabolites. The in vitro MTT assay revealed the cytotoxic trait of these assemblies, a finding that was substantiated by in vivo assays employing the Caenorhabditis elegans (C. elegans) model, which revealed that the toxic effects were more pronounced and dose-specific in the case of metabolites that had aged via longer preincubation. We hence report a compelling phenomenon wherein these metabolites not only aggregate but transform into a soft, ordered assembly over time, eventually crystallizing upon extended incubation, leading to pathological implications. Our study suggests that the amyloidogenic nature of the involved metabolites could be a common etiological link in IEMs, potentially providing a unified perspective to study their pathophysiology, thus offering exciting insights into the development of targeted interventions for these metabolic disorders.

Designed Local Electric Fields-Promising Tools for Enzyme Engineering.

Designing efficient catalysts is one of the ultimate goals of chemists. In this Perspective, we discuss how local electric fields (LEFs) can be exploited to improve the catalytic performance of supramolecular catalysts, such as enzymes. More specifically, this Perspective starts by laying out the fundamentals of how local electric fields affect chemical reactivity and review the computational tools available to study electric fields in various settings. Subsequently, the advances made so far in optimizing enzymatic electric fields through targeted mutations are discussed critically and concisely. The Perspective ends with an outlook on some anticipated evolutions of the field in the near future. Among others, we offer some pointers on how the recent data science/machine learning revolution, engulfing all science disciplines, could potentially provide robust and principled tools to facilitate rapid inference of electric field effects, as well as the translation between optimal electrostatic environments and corresponding chemical modifications.

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction.

Understanding structure-function relationships in proteins is pivotal in their development as industrial biocatalysts. In this regard, rational engineering of protein active site access pathways and various tunnels and channels plays a central role in designing competent enzymes with high stability and enhanced efficiency. Here, we report the rational evolution of a thermostable cytochrome P450, CYP175A1, to catalyze the C-H activation reaction of longer-chain alkanes. A strategy combining computational tools with experiments has shown that the substrate scope and enzymatic activity can be enhanced by rational engineering of certain important channels such as the substrate entry and water channels along with the active site of the enzyme. The evolved enzymes showed an improved catalytic rate for hexadecane hydroxylation with high regioselectivity. The Q67L/Y68F mutation showed binding of the substrate in the active site, water channel mutation L80F/V220T showed improved catalytic activity through the peroxide shunt pathway and substrate entry channel mutation W269F/I270A showed better substrate accessibility to the active pocket. All-atom MD simulations provided the rationale for the inactivity of the wild-type CYP175A1 for hexadecane hydroxylation and predicted the above hot-spot residues to enhance the activity. The reaction mechanism was studied by QM/MM calculations for enzyme-substrate complexes and reaction intermediates. Detailed thermal and thermodynamic stability of all the mutants were analyzed and the results showed that the evolved enzymes were thermally stable. The present strategy showed promising results, and insights gained from this work can be applied to the general enzymatic system to expand substrate scope and improve catalytic activity.

Tyr 118-Mediated Electron Transfer is Key to the Chlorite Decomposition in Heme-Dependent Chlorite Dismutase.

Chlorite dismutase (Cld) is a crucial enzyme that catalyzes the decomposition of chlorite ions into chloride ions (Cl-) and molecular oxygen (O2). Despite playing an important role in the detoxification of toxic chlorite ions, the mechanism of cleavage of the Cl-O bond by Cld remains highly debatable. The present study highlights the mechanism of such Cl-O bond cleavage in Cld using sophisticated computational tools such as hybrid quantum mechanical/molecular mechanical calculations and long-time scale molecular dynamics simulations. Here, we show that Cld forms a high spin ferric hexacoordinated substrate adduct in the presence of a chlorite ion, which subsequently reduces to a ferrous state. Our study shows a stepwise pathway with the homolytic cleavage of the Cl-O bond that produces a high spin Fe(III)-OH species and a diradicaloid species formed by the combination of a chlorine-based ClO• radical and a protein-based tyrosine118• radical. The findings provide significant insights into Cl-O bond cleavage and O2 formation which shows a crucial role of the tyrosine118 during the electron transfer process.

A porphyrin-based molecular cage guided by designed local-electric field is highly selective and efficient.

The present work outlines a general methodology for designing efficient catalytic machineries that can easily be tweaked to meet the demands of the target reactions. This work utilizes a principle of the designed local electric field (LEF) as the driver for an efficient catalyst. It is demonstrated that by tweaking the LEF, we can catalyze the desired hydroxylation products with enantioselectivity that can be changed at will. Using computation tools, we caged a synthetic analog of heme porphyrin (HM1) and investigated the pharmaceutically relevant conversion of tetralin to tetralol, inside the modified supramolecular cage. The QM/MM calculations demonstrate a resulting catalytic efficiency with virtually absolute R-selectivity for the tetralin hydroxylation. Our calculations show that the LEF of the supramolecular cage and HM1 exert a strong electric field along the Fe-O reaction axis, which is the main driving force for enhanced reactivity. At the same time, the supramolecular cage applies a lateral LEF that regulates the enantioselectivity. We further demonstrate that swapping the charged/polar substitution in the supramolecular cage switches the lateral LEF which changes the enantioselectivity of hydroxylation from R to S.

Electro-Freezing of Supercooled Water Is Induced by Hydrated Al3+ and Mg2+ Ions: Experimental and Theoretical Studies.

This work reports that the octahedral hydrated Al3+ and Mg2+ ions operate within electrolytic cells as kosmotropic (long-range order-making) "ice makers" of supercooled water (SCW). 10-5 M solutions of hydrated Al3+ and Mg2+ ions each trigger, near the cathode (-20 ± 5 V), electro-freezing of SCW at -4 °C. The hydrated Al3+ ions do so with 100% efficiency, whereas the Mg2+ ions induce icing with 40% efficiency. In contrast, hydrated Na+ ions, under the same experimental conditions, do not induce icing differently than pure water. As such, our study shows that the role played by Al3+ and Mg2+ ions in water electro-freezing is impacted by two synchronous effects: (1) a geometric effect due to the octahedral packing of the coordinated water molecules around the metallic ions, and (2) the degree of polarization which these two ions induce and thereby acidify the coordinated water molecules, which in turn imparts them with an ice-like structure. Long-duration molecular dynamics (MD) simulations of the Al3+ and Mg2+ indeed reveal the formation of "ice-like" hexagons in the vicinity of these ions. Furthermore, the MD shows that these hexagons and the electric fields of the coordinate water molecules give rise to ultimate icing. As such, the MD simulations provide a rational explanation for the order-making properties of these ions during electro-freezing.

Design and Synthesis of Co-initiators via Base-Catalysed Sequential Conjugate Addition: Application in Photoinduced Radical Polymerisation Reaction.

Applications of photochemistry are becoming very popular in modern-day life due to its operational simplicity, environmentally friendly and economically sustainable nature in comparison to thermochemistry. In particular photoinduced radical polymerisation (PRP) reactions are finding more biological applications and especially in the areas of dental restoration processes, tissue engineering and artificial bone generation. A type-II photoinitiator and co-initiator-promoted PRP turned out to be a cost-effective protocol, and herein we report the design and synthesis of a new efficient co-initiator for a PRP reaction via a barrierless sequential conjugate addition reaction. Experimental mechanistic observations have been further complemented by computational data. Time for newly synthesised 1,2-benzenedithiol (DTH) based co-initiator promoted polymerisation of urethane dimethacrylate (UDMA, 70 %) and triethylene glycol dimethacrylate (TEGDMA, 30 %) in presence of 450 nm LED (15 W) under the aerobic conditions is 38 seconds. Polymeric material has high glass transition temperature, improved mechanical strength (860 BHN) and longer in-depth polymerisation (3 cm).

A single site mutation can induce functional promiscuity in homoserine kinase.

L-Homoserine kinase is crucial in the biosynthesis of L-threonine, L-isoleucine, and L-methionine, where it catalyzes ATP-dependent phosphorylation of L-homoserine (Hse) to yield L-homoserine phosphate as its native activity. However, a single site mutation of H138 → L shows the emergence of ATPase activity as a secondary function. However, a previous mechanistic study proposes direct involvement of ATP and the substrate without any catalytic base; therefore, how the mutation of H138 → L causes the secondary function remains an enigma. Using computational tools herein, we provide new insight into the catalytic mechanism of L-homoserine kinase, showing direct involvement of H138 as a catalytic base. We show that mutation of H138 → L opens a new water channel connecting ATP, which facilitates the ATPase activity and reduces the native activity. The proposed mechanism agrees with the experimental finding that an H138 → L mutation reduces the kinase activity but enhances a promiscuous function, i.e. ATPase activity. Since homoserine kinase catalyzes the biosynthesis of amino acids, we believe that an accurate mechanism could be significant for enzyme engineering to synthesize amino acid analogs.

Substrate Conformation Regulates Aromatic C-H Vs C-F Bond Activation in Heme-Dependent Tyrosine Hydroxylase.

A recently discovered heme-dependent enzyme tyrosine hydroxylase (TyrH) offers a green approach for functionalizing the high-strength C-H and C-F bonds in aromatic compounds. However, there is ambiguity regarding the nature of the oxidant (compound 0 or compound I) involved in activating these bonds. Herein, using comprehensive molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical calculations, we reveal that it is compound I (Cpd I) that acts as the primary oxidant involved in the functionalization of both C-F and C-H bonds. The energy barrier for C-H and C-F activation using compound 0 (Cpd 0) as an oxidant was very high, indicating that Cpd 0 cannot be an oxidant. Consistent with the previous experimental finding, our simulation shows two different conformations of the substrate, where one orientation favors the C-H activation, while the other conformation prefers the C-F activation. As such, our mechanistic study shows that nature utilizes just one oxidant, that is, Cpd I, but it is the active site conformation that decides whether it selects C-F or C-H functionalization which may resemble involvement of two different oxidants.

Decarboxylation and Protonation Enigma in the H85Q Mutant of Cytochrome P450OleT.

Cytochrome P450OleT (CYP450OleT), a member of CYP450 peroxygenases, catalyzes unusual decarboxylation activity. Unlike other members of the peroxygenases family, CYP450OleT possesses a histidine at the 85th position, which was supposed to be the root cause of the decarboxylation activity in CYP450OleT. This work addresses the His85 → Gln mutant paradox, where mutation of His → Gln still shows efficient decarboxylation activity in CYP450OleT. The MD simulation of the H85Q mutant of CYP450OleT shows that in the absence of the histidine at the 85th position, an Asp239 plays a similar role via a well-organized water channel. Our simulation shows that such a water channel is vital for the optimal substrate positioning needed for the decarboxylation activity and is gated by the Q85-N242 residue pair. Interestingly, the MD simulation of the WT CYP450BSß shows a closed channel that blocks access to the Glu236 (analogous residue to Asp239 in CYP450OleT), and therefore, CYP450BSß shows low decarboxylation activity.

Development of indolealkylamine derivatives as potential multi-target agents for COVID-19 treatment.

COVID-19 is a complex disease with short-term and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. As many drugs targeting single targets showed only limited effectiveness against COVID-19, here, we aimed to explore a multi-target strategy. We synthesized a focused compound library based on C2-substituted indolealkylamines (tryptamines and 5-hydroxytryptamines) with activity for three potential COVID-19-related proteins, namely melatonin receptors, calmodulin and human angiotensin converting enzyme 2 (hACE2). Two molecules from the library, 5e and h, exhibit affinities in the high nanomolar range for melatonin receptors, inhibit the calmodulin-dependent calmodulin kinase II activity and the interaction of the SARS-CoV-2 Spike protein with hACE2 at micromolar concentrations. Both compounds inhibit SARS-CoV-2 entry into host cells and 5h decreases SARS-CoV-2 replication and MPro enzyme activity in addition. In conclusion, we provide a proof-of-concept for the successful design of multi-target compounds based on the tryptamine scaffold. Optimization of these preliminary hit compounds could potentially provide drug candidates to treat COVID-19 and other coronavirus diseases.

How Can Static and Oscillating Electric Fields Serve in Decomposing Alzheimer's and Other Senile Plaques?

Alzheimer's disease is one of the most common neurodegenerative conditions, which are ascribed to extracellular accumulation of ß-amyloid peptides into plaques. This phenomenon seems to typify other related neurodegenerative diseases. The present study uses classical molecular-dynamics simulations to decipher the aggregation-disintegration behavior of ß-amyloid peptide plaques in the presence of static and oscillating oriented external electric fields (OEEFs). A long-term disintegration of such plaques is highly desirable since this may improve the prospects of therapeutic treatments of Alzheimer's disease and of other neurodegenerative diseases typified by senile plaques. Our study illustrates the spontaneous aggregation of the ß-amyloid, its prevention and breakdown when OEEF is applied, and the fate of the broken aggregate when the OEEF is removed. Notably, we demonstrate that the usage of an oscillating OEEF on ß-amyloid aggregates appears to lead to an irreversible disintegration. Insight is provided into the root causes of the various modes of aggregation, as well as into the different fates of OEEF-induced disintegration in oscillating vs static fields. Finally, our simulation results are compared to the well-established TTFields and the Deep Brain Stimulation (DBS) therapies, which are currently used options for treatments of Alzheimer's disease and other related neurodegenerative diseases.

Unraveling topoisomerase IA gate dynamics in presence of PPEF and its preclinical evaluation against multidrug-resistant pathogens.

Type IA topoisomerases maintain DNA topology by cleaving ssDNA and relaxing negative supercoils. The inhibition of its activity in bacteria prevents the relaxation of negative supercoils, which in turn impedes DNA metabolic processes leading to cell death. Using this hypothesis, two bisbenzimidazoles, PPEF and BPVF are synthesized, selectively inhibiting bacterial TopoIA and TopoIII. PPEF stabilizes the topoisomerase and topoisomerase-ssDNA complex, acts as an interfacial inhibitor. PPEF display high efficacy against ~455 multi-drug resistant gram positive and negative bacteria. To understand molecular mechanism of inhibition of TopoIA and PPEF, accelerated MD simulation is carried out, and results suggested that PPEF binds, stabilizes the closed conformation of TopoIA with -6Kcal/mol binding energy and destabilizes the binding of ssDNA. The TopoIA gate dynamics model can be used as a tool to screen TopoIA inhibitors as therapeutic candidates. PPEF and BPVF cause cellular filamentation and DNA fragmentation leading to bacterial cell death. PPEF and BPVF show potent efficacy against systemic and neutropenic mouse models harboring E. coli, VRSA, and MRSA infection without cellular toxicity.

Local Electric Fields: From Enzyme Catalysis to Synthetic Catalyst Design.

This Mini-Review Article outlines recent advances in the study of local electric field (LEF) governed enzyme catalysis and the application of the LEF principle in synthetic catalyst design. We start by discussing the electrostatics principles that drive enzyme catalysis, and its experimental verifications through vibrational Stark spectroscopy. Subsequently, we describe aspects of LEFs other than catalysis, i.e., induction of mechanistic crossovers, among others. Here, we focus on the early work done using computational tools, along with some recent contributions. Following an in-depth discussion of the role of LEFs in enzyme catalysis, we then highlight some recent works on designed local electric fields (D-LEF) and their applications in organic synthesis. Subsequently, we turn to D-LEFs in synthetic enzymes and supramolecular systems (cf. the work by the Head-Gordon group). We end by discussing some of the software packages that have been developed to analyze local electric fields computationally. Overall, the present Mini-Review Article paints an insightful picture of the current state of the art using LEF in enzyme catalysis and its application for further bioengineering and synthetic organic frameworks in a broad perspective.

Unraveling key interactions and the mechanism of demethylation during hAGT-mediated DNA repair via simulations.

Alkylating agents pose the biggest threat to the genomic integrity of cells by damaging DNA bases through regular alkylation. Such damages are repaired by several automated types of machinery inside the cell. O6-alkylguanine-DNA alkyltransferase (AGT) is an enzyme that performs the direct repair of an alkylated guanine base by transferring the alkyl group to a cysteine residue. In the present study, using extensive MD simulations and hybrid QM/MM calculations, we have investigated the key interactions between the DNA lesion and the hAGT enzyme and elucidated the mechanisms of the demethylation of the guanine base. Our simulation shows that the DNA lesion is electrostatically stabilized by the enzyme and the Arg135 of hAGT enzyme provides the main driving force to flip the damaged base into the enzyme. The QM/MM calculations show demethylation of the damaged base as a three-step process in a thermodynamically feasible and irreversible manner. Our calculations show that the final product forms via Tyr114 in a facile way in contrast to the previously proposed Lys-mediated route.

Computations reveal a crucial role of an aromatic dyad in the catalytic function of plant cytochrome P450 mint superfamily.

Enzymes are highly specific for their native functions, however with advances in bioengineering tools such as directed evolution, several enzymes are being repurposed for the secondary function of contemporary significance(Khersonsky and Tawfik, 2010 [1]). Due to the functional versatility, the Cytochrome P450 (CYP450) superfamily has become the ideal scaffold for such bioengineering. In the current study, using MD (molecular dynamics) simulations and hybrid QM/MM (Quantum mechanics/molecular mechanics) calculations, we have studied the mechanism of spontaneous emergence of a secondary function due to a single site mutation in two plant CYP450 enzymes from the mint family. The MD simulations of WT (wild type) CYP71D18 and CYP71D13 enzymes and their variants show a crucial gating mechanism by aromatic dyad formed by Phe121 and Phe363 which regulates the substrate recognition. The QM/MM calculations reveal that the hydroxylation reactions at C3 and C6 positions in WT CYP71D18 and CYP71D13 enzymes as well as their variants follow a hydrogen atom transfer (HAT) followed by a single electron transfer (SET) mechanism, which is different from the typical rebound mechanism shown by most of the CYP450 enzymes.

The Performance of Different Water Models on the Structure and Function of Cytochrome P450 Enzymes.

Modeling approaches and modern simulations to investigate the biomolecular structure and function rely on various methods. Since water molecules play a crucial role in all sorts of chemistry, the accurate modeling of water molecules is vital for such simulations. In cytochrome P450 (CYP450), in particular, water molecules play a key role in forming active oxidant that ultimately performs oxidation and metabolism. In the present study, we have highlighted the behavior of the three most widely used water models─TIP3P, SPC/E, and OPC─for three different CYP450 enzymes─CYP450BM3, CYP450OleT, and CYP450BSß─during MD simulations and QM/MM calculations. We studied the various properties, such as RMSD, RMSF, H-bond, water occupancy, and hydrogen atom transfer (HAT), using QM/MM calculations and compared them for all three water models. Our study shows that the stabilities of the enzyme complexes are well maintained in all three water models. However, the OPC water model performs well for the polar active sites, that is, in CYP450OleT and CYP450BSß, while the TIP3P water model is superior for the hydrophobic site, such as CYP450BM3.

Regulation of Tyrosinase Enzyme Activity by Glutathione Peroxidase Mimics.

Hydrogen peroxide plays a crucial role in the melanogenesis process by regulating the activity of the key melanin-forming enzyme tyrosinase, responsible for the browning of fruits, vegetables, and seafood. Therefore, a molecule with dual activities, both efficient tyrosinase inhibition and strong hydrogen peroxide degrading ability, may act as a promising antibrowning agent. Herein, we report highly efficient selone-based mushroom tyrosinase inhibitors 2 and 3 with remarkable glutathione peroxidase (GPx) enzyme-like activity. The presence of benzimidazole moiety enhances the tyrosinase inhibition efficiency of selone 2 (IC50 = 0.4 µM) by almost 600 times higher than imidazole-based selone 1 (IC50 = 238 µM). Interestingly, the addition of another aromatic ring to the benzimidazole moiety has led to the development of an efficient lipid-soluble tyrosinase inhibitor 3 (IC50 = 2.4 µM). The selenium center and the -NH group of 2 and 3 are extremely crucial to exhibit high GPx-like activity and tyrosinase inhibition potency. The hydrophobic moiety of the inhibitors (2 and 3) further assists them in tightly binding at the active site of the enzyme and facilitates the C═Se group to strongly coordinate with the copper ions. Inhibitor 2 exhibited excellent antibrowning and polyphenol oxidase inhibition properties in banana and apple juice extracts.