Semirational Design Strategy To Enhance the Thermostability and Catalytic Activity of Cytochrome P450 105D7 for the Degradation of the Pharmaceutically Active Compounds: Diclofenac.

Pharmaceutically active compounds are an important category of emerging pollutants, and their biological transformation processes in the environment are crucial for understanding and evaluating the migration, transformation, and environmental fate of emerging pollutants. The cytochrome P450 105 enzyme family has been proven to play an important role in the degradation of exogenous environmental pollutants. However, its thermostability and catalytic activity still need to be improved to better adapt to complex environmental conditions. This work elucidates the key mechanisms and important residues of the degradation reaction through multiple computational strategies, establishes a mutation library, and obtains 21 single-point mutation designs. Experimental verification showed that 16 single mutants had enhanced thermostability, with the R89F and L197Y mutants showing the highest increases in thermostability at 135 and 119% relative to the wild-type enzyme, respectively. Additionally, as a result of the higher specific activity of D390Q, it was selected for combination mutagenesis, ultimately resulting in three combination mutants (R89F/L197Y, R89F/D390Q, and R89F/L197Y/D390Q) with enhanced thermostability and catalytic activity. This study provides a modification approach for constructing efficient enzyme variants through semirational design and can contribute to the development of control technologies for emerging pollutants.

Stereoselective Construction of ß-, γ-, and δ-Lactam Rings via Enzymatic C-H Amidation.

Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Methods for the asymmetric synthesis of these molecules are therefore highly desirable, particularly through the selective functionalization of unreactive aliphatic C-H bonds. Here we show the development of a strategy for the asymmetric synthesis of ß-, γ-, and δ-lactams via hemoprotein-catalysed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation yielding the desired lactam products in high yields, high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Additionally, an alkaloid natural product and a drug molecule were synthesized chemoenzymatically in much fewer steps (7-8 vs. 11-12) than previously reported, further demonstrating the power of biosynthetic strategy for the preparation of complex bioactive molecules.

Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with 'rule-of-three' properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Stereoselective Construction of ß-, γ-, and δ-Lactam Rings via Enzymatic C-H Amidation.

Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Given their widespread presence in bioactive molecules, methods for the asymmetric synthesis of these molecules, in particular through the selective functionalization of ubiquitous yet unreactive aliphatic C-H bonds, are highly desirable. In this study, we report the development of a novel strategy for the asymmetric synthesis of 4-, 5-, and 6-membered lactams via an unprecedented hemoprotein-catalyzed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation producing an array of ß-, γ-, and δ-lactam molecules in high yields, with high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps in these reactions and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Using this system, it was possible to accomplish the chemoenzymatic total synthesis of an alkaloid natural product and a drug molecule in much fewer steps (7-8 vs. 11-12) than previously possible, which showcases the power of this biosynthetic strategy toward enabling the preparation of complex bioactive molecules.

Computational Exploration of Anomalous Regioselectivities in Cycloadditions of Ketenes to Oxazolines.

Thermal (2 + 2) cycloadditions of several N-carboalkoxy (R)-2-tert-butyldihydrooxazoles with ketenes have been studied experimentally by the Ghosez group. Contrary to results from Seebach and co-workers that the electrophilic addition of acylating agents occurs ß to dihydrooxazole nitrogen, Ghosez found major cycloadducts resulting from an attack of ketene carbonyl carbon ß to oxygen. We investigate the potential energy surface for the cycloaddition of diphenyl- and phenylchloroketenes to two (R)-2-tert-butyldihydrooxazoles with ωB97X-D and mPW1PW91 density functional theory and DLPNO-CCSD(T) wave function theory. These (2 + 2) cycloadditions are concerted but highly asynchronous, and the selectivity trends in ketene addition cases are in good agreement with the experiment. We propose a model based on the buildup of charge in oxazoline to reconcile the regiochemical differences between Ghosez and Seebach's observations.

An Enzymatic Platform for Primary Amination of 1-Aryl-2-alkyl Alkynes.

Propargyl amines are versatile synthetic intermediates with numerous applications in the pharmaceutical industry. An attractive strategy for efficient preparation of these compounds is nitrene propargylic C(sp3)-H insertion. However, achieving this reaction with good chemo-, regio-, and enantioselective control has proven to be challenging. Here, we report an enzymatic platform for the enantioselective propargylic amination of alkynes using a hydroxylamine derivative as the nitrene precursor. Cytochrome P450 variant PA-G8 catalyzing this transformation was identified after eight rounds of directed evolution. A variety of 1-aryl-2-alkyl alkynes are accepted by PA-G8, including those bearing heteroaromatic rings. This biocatalytic process is efficient and selective (up to 2610 total turnover number (TTN) and 96% ee) and can be performed on preparative scale.

Metabolic activation mechanism of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB136) by cytochrome P450 2B6: A QM/MM approach.

Cytochrome P450 enzymes (CYPs) play an essential role in the bio-transformation of polychlorinated biphenyls (PCBs). The present work implemented quantum mechanic/molecular mechanic methods (QM/MM) and density functional theory (DFT) to study the metabolic activation of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB136) catalyzed by CYP2B6. Electrophilic additions at the Cα and Cß positions generate different active intermediates. The electrophilic addition energy barrier of Cß is 10.9 kcal/mol higher than that of Cα, and Cα is the preferred site for the electrophilic addition reaction. Based on the previous experimental studies, this work investigated the mechanism of converting active intermediates into OH-PCB136, which has high toxicity in a non-enzymatic environment. Structural analysis via the electrostatic and noncovalent interactions indicates that Phe108, Ile114, Phe115, Phe206, Phe297, Ala298, Leu363, Val367, TIP32475 and TIP32667 play crucial roles in substrate recognition and metabolism. The analysis suggests that the halogen-π interactions are important factors for the metabolism of CYP2B6 to halogenated environmental pollutants. This work improved the understanding of the metabolism and activation process of chiral PCBs, and can be used as a guide to improve the microbial degradation efficiency of PCB136.

Computational study on the metabolic activation mechanism of PeCDD by Cytochrome P450 1A1.

Cytochrome P450 enzymes (CYPs) are crucial for metabolizing dioxin compounds such as 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD). Here we have applied molecular dynamic simulations (MD), quantum mechanics/molecular mechanics methods (QM/MM) and density functional theory (DFT) to investigate the metabolic activation and transformation of PeCDD catalyzed by CYP1A1. Our QM/MM calculations highlight that PeCDD can be activated by P450s through the well-known electrophilic addition mechanism with an average energy barrier of 20.9 kcal/mol. Based on the results of previous experimental studies, further conversions of ketone products and epoxidation products that are mediated by P450 enzymes were investigated through DFT calculations. Analysis of the structures via the noncovalent interactions (NCI) method and the distortion-interaction model suggests that amino acids Ser122, Ala317, Ile386 and Leu496 play important roles in the metabolic process.

Degradation mechanism of biphenyl and 4-4'-dichlorobiphenyl cis-dihydroxylation by non-heme 2,3 dioxygenases BphA: A QM/MM approach.

Biphenyl 2,3-dioxygenase (BphA), a Rieske-type and first enzyme in the aerobic degradation process, plays a key role in the metabolizing process of biphenyl/polychlorinated biphenyl aromatic pollutants in the environment. To understand the catalytic mechanism of biphenyl 2,3-dioxygenase, the conversions leading to the cis-diols are investigated by means of quantum mechanics/molecular mechanics (QM/MM) method. A hydroperoxo-iron (III) species is involved in the enzyme-catalyzed reaction. Herein, we explored the direct reaction mechanism of hydroperoxo-iron (III) species with biphenyl and 4-4'-dichlorobiphenyl. The reaction process involves an epoxide intermediate, it could develop into a carbocation intermediate, and ultimately evolve into a cis-diol product. The important roles of several residues during the dioxygenation process were highlighted. This study may provide theoretical support for further directed mutations and enzymatic engineering of BphA, as well as promote the development of degrading environmentally persistent biphenyl/polychlorinated biphenyl aromatic contaminants.

Experimental Investigation on the Nonlinear Coupled Flutter Motion of a Typical Flat Closed-Box Bridge Deck.

The nonlinear post-flutter instabilities were experimentally investigated through two-degree-of-freedom sectional model tests on a typical flat closed-box bridge deck (width-to-depth ratio 9.14). Laser displacement sensors and piezoelectric force balances were used in the synchronous measurement of dynamic displacement and aerodynamic force. Beyond linear flutter boundary, the sectional model exhibited heave-torsion coupled limit cycle oscillation (LCOs) with an unrestricted increase of stable amplitudes with reduced velocity. The post-critical LCOs vibrated in a complex mode with amplitude-dependent mode modulus and phase angle. Obvious heaving static deformation was found to be coupled with the large-amplitude post-critical LCOs, for which classical quasi-steady theory was not applicable. The aerodynamic torsional moment and lift during post-critical LCOs were measured through a novel wind-tunnel technique by 4 piezoelectric force balances. The measured force signals were found to contain significantly higher-order components. The energy evolution mechanism during post-critical LCOs was revealed via the hysteresis loops of the measured force signals.

Degradation mechanism for Zearalenone ring-cleavage by Zearalenone hydrolase RmZHD: A QM/MM study.

The danger of zearalenone (ZEN) as an endocrine disruptor to humans and the environment has aroused increasing attention. In this study, we implemented the quantum mechanics/molecular mechanics (QM/MM) method to investigate the degradation mechanism of ZEN hydrolase (RmZHD) toward ZEN at the atomic level. The degradation process involves two concerted reaction pathways, where the active site contains a Ser-His-Glu triplet as a proton donor. With the Boltzmann-weighted average potential barriers of 18.1 and 21.5 kcal/mol, the process undergoes proton transfer and nucleophilic-substituted ring opening to form a hydroxyl product. Non-covalent interaction analyses elucidated hydrogen bonding between key amino acids with ZEN. The electrostatic influence analysis of 16 amino acids proposes residues Asp34 and His128 as the possible mutation target for future mutation design of enzyme RmZHD. An in-depth investigation of the protein environment of RmZHD can improve the bioremediation efficiency of endocrine disrupting chemicals.

Biodegradation mechanism of polyesters by hydrolase from Rhodopseudomonas palustris: An in silico approach.

Massively used plastics have caused worldwide environmental concerns. Polyesters like polylactic acid (PLA) are one of the mostly used plastics due to its excellent physical and chemical properties and low-cost advantages. It is critical to develop the elimination and recycle techniques for polyesters. Experimental studies have shown that a hydrolase RPA1511 isolated from Rhodopseudomonas palustris can efficiently depolymerize polylactic acid (PLA) into oligomers and monomers. It was also active against emulsified aliphatic polymers as well as multipurpose soluble ester monomers (α-naphthyl ester and p-nitrophenyl ester). In the present study, molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area method were applied to screen all amino acids from hydrolase RPA1511 and identify the most important amino acids during substrate binding. Seven substrates were considered: PLA (dimer and tetramer), polycaprolactone, butylene succinate, 1-naphthyl acetate, 2-naphthyl formate, p-nitrophenyl acetate. The results highlighted the importance of amino acids like Tyr139, Tyr213, Arg259, Thr46. Subsequent quantum mechanics/molecular mechanics calculations were also performed to determine the detailed degradation mechanism of hydrolase RPA1511 toward PLA and explore the role of the active site residues during catalysis. The results demonstrated that degradation involves two elementary steps: enzyme acylation and PLA hydrolysis. The corresponding Boltzmann average barriers are 20.40 kcal/mol and 14.45 kcal/mol. The electrostatic influence analysis of 15 amino acids on the rate-determining step indicated that amino acids His114, Trp219 and Ala273 facilitate the reaction while the Arg244 suppresses the reaction which may serve as future mutation studies to enhance the enzymatic efficiency.

Catalytic Mechanism for 2,3-Dihydroxybiphenyl Ring Cleavage by Nonheme Extradiol Dioxygenases BphC: Insights from QM/MM Analysis.

An extradiol-cleaving catecholic dioxygenase, 2,3-dihydroxybiphenyl dioxygenase, plays important roles in the catabolism of biphenyl/polychlorinated biphenyl aromatic contaminants in the environment. To better elucidate the biodegradable pathway, a theoretical investigation of the ring-opening degradation of 2,3-dihydroxybiphenyl (DHBP) was performed with the aid of quantum mechanical/molecular mechanical calculations. A quintet state of the DHBP-iron-dioxygen group adducts was found to be the reactive state with a substrate radical-FeII-superoxo (DHBP•↑-FeII-O2•-↓) character. The HOO• species was the reactive oxygen species responsible for the subsequent attack of DHBP. Among the whole reaction energy profile, the first step in proton-coupled electron transfer was determined to be the rate-determining step with a potential energy barrier of 17.2 kcal/mol, which is close to the experimental value (14.7 kcal/mol). Importantly, the residue His194 shows distinct roles in the catalytic cycle, where it acts as an acid-base catalyst to deprotonate the hydroxyl group of DHBP at an early stage, then stabilizes the negative charge on the dioxygen group, and, at the final stage, promotes the semialdehyde product formation as a proton donor.

Computational evidence for the degradation mechanism of haloalkane dehalogenase LinB and mutants of Leu248 to 1-chlorobutane.

The catalytic degradation ability of the haloalkane dehalogenase LinB toward 1-chlorobutane (1-CB) was studied using a combined quantum mechanics/molecular mechanics (QM/MM) approach. Two major processes are involved in the LinB-catalyzed removal of halogens: dechlorination and hydrolyzation. The present study confirmed the experimentally proposed reaction path at the molecular level. Moreover, based on nucleophilic substitution mechanism (SN2 reaction), dechlorination was found to be the rate-determining step of the entire reaction process. In this study, the Boltzmann-weighted average barrier for dechlorination was determined to be 17.0 kcal mol-1, which is fairly close to the experimental value (17.4 kcal mol-1). The state of His107 and the influence of Leu248 on the dechlorination process were also explored. In addition, an intriguing phenomenon was discovered: the potential energy barrier decreased by 7.5 kcal mol-1 when the Leu248 residue was mutated into Phe248. This discovery might be of great help to design new mutant enzymes or novel biocatalysts.

QM/MM study of the reaction mechanism of Cl-cis,cis-muconate with muconate lactonizing enzyme.

The lactonization process of Cl-cis,cis-muconate catalyzed by anti-muconate lactonizing enzyme (anti-MLE) was studied theoretically with the aid of a combined quantum mechanics/molecular mechanics (QM/MM) approach. Two elementary processes steps involved in the lactanization process were investigated. The calculated energy barriers agree well with the experimental values. The present work provided the explicit structures of the enolate anion intermediates. The electrostatic influence analysis highlighted residues Arg51, Gln294 and TIP383 for the MLE-Cl-2 system and the residue Asn193 for the MLE-Cl-4 system as the possible mutation targets for rational design of anti-MLE in future enzyme modification.

The growth mechanism of polycyclic aromatic hydrocarbons from the reactions of anthracene and phenanthrene with cyclopentadienyl and indenyl.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, mutagenic and/or carcinogenic to humans. To reduce the emission of PAHs, it's significant and indispensable to explore the PAH formation mechanism. In the present work, the growth mechanism of PAHs from the reactions of anthracene and phenanthrene with cyclopentadienyl and indenyl radicals was investigated with the aid of high-accuracy quantum chemistry calculation. The rate constants of key elementary steps were calculated by meaning of the canonical variation transition-state (CVT) theory with the small curvature tunneling (SCT) correction over the temperature range of 400-1400 K. The mechanism of the PAH formation involves in six elementary steps, addition reaction, ring closure, intramolecular H-shift, cleavage of CC bond, intramolecular H-shift and unimolecular elimination of CH3 or H. The cleavage of CC bond is the rate-determining step due to the high barrier. The formation of PAHs from the reactions of anthracene with cyclopentadienyl and indenyl radicals is easier than that from the reactions of phenanthrene.

Adsorption of bisphenol A to a carbon nanotube reduced its endocrine disrupting effect in mice male offspring.

Soluble carbon nanotubes (CNTs) have shown promise as materials for adsorption of environmental contaminants such as Bisphenol A (BPA), due to the high adsorption capacity and strong desorption hysteresis of BPA on CNTs. The adsorption of BPA to CNTs may change the properties of both BPA and CNTs, and induce different toxicity to human and living systems from that of BPA and CNTs alone. Herein, we report that oral exposure of BPA/MWCNT-COOH (carboxylated multi-walled carbon nantubes) adduct to mice during gestation and lactation period decreased the male offspring reproductive toxicity compared with those induced by BPA alone. The adduct decreased malondialdehyde (MDA) level in testis and follicle-stimulating hormone (FSH) in serum, but increased the level of serum testosterone in male offspring in comparison to BPA alone. Our investigations broadened the knowledge of nanotoxicity and provided important information on the safe application of CNTs.

Atmospheric oxidation mechanism and kinetic studies for OH and NO3 radical-initiated reaction of methyl methacrylate.

The mechanism for OH and NO3 radical-initiated oxidation reactions of methyl methacrylate (MMA) was investigated by using density functional theory (DFT) molecular orbital theory. Geometrical parameters of the reactants, intermediates, transition states, and products were fully optimized at the B3LYP/6-31G(d,p) level. Detailed oxidation pathways were presented and discussed. The rate constants were deduced by the canonical variational transition-state (CVT) theory with the small-curvature tunneling (SCT) correction and the multichannel Rice-Ramspergere-Kassele-Marcus (RRKM) theory, based on the potential energy surface profiles over the general atmospheric temperature range of 180-370 K. The calculated results were in reasonable agreement with experimental measurement.