ABSTRACT Multi‐subunit formate dehydrogenases (FDHs), which catalyze the interconversion of formate and carbon dioxide (CO 2 ), have drawn increasing attention for mitigating climate change and advancing environmental protection owing to their advantages of oxygen tolerance and easy heterogenous expression. However, differently sourced multi‐subunit FDHs exhibit distinct catalytic biases, and the reasons remain unclear. On the basis of the exceptional observation of Rhodobacter aestuarii FDH favoring CO 2 reduction, this study unveiled an oxidation inhibition effect in exclusively NADH/NAD + ‐involved catalysis via kinetics analysis in terms of different redox couples. Substrate truncation positioned Fdhβ as the predominant subunit. Further studies based on structural and electrochemical insights interpreted that the slow desorption of NADH is the underlying determinant for the apparent catalytic bias. Knowledge‐based rational design helped obtain a beneficial variant, Ra FDH β E260Y, with a 10‐fold increased catalytic activity in CO 2 reduction, highlighting its potential for CO 2 biotransformation and applications in low‐carbon biomanufacturing. Eventually, bioinformatic analysis suggested that the diaphorase‐like subunits and the catalysis regulation mechanism may widely exist in living organisms for modulating the redox balance of oxidoreductases, providing new insights into metabolism and catabolism.

Strictosidine synthase (STR) is an enzyme catalyzing the Pictet-Spengler (PS) reaction between tryptamine and secologanin to generate strictosidine. The product is a key intermediate in the biosynthesis of numerous indole alkaloids in plants, many of which exhibit significant therapeutic potential. Recent studies have shown that STR displays promiscuous activity in catalyzing reactions of tryptamine analogs to form new alkaloid skeletons. Notably, the STR from Rauvolfia serpentina (RsSTR) has recently been demonstrated to synthesize strictosidine analogs with an uncommon piperazinyl-[1,2-a] indole scaffold from 1H-indole-1-ethanamine (1-IEA) and secologanin. In this study, by using quantum chemical calculations, the detailed mechanism of the RsSTR-catalyzed reaction between 1-IEA and secologanin was revealed at the atomic level. The structures of all intermediates and transition states involved in the reaction pathway were optimized, and the energy profile was obtained. Interestingly, it was found that the rate-determining step changes from proton transfer in the natural reaction to cyclization here. Comparative analysis revealed that this shift is attributed to the different interactions between the substrate and active site residues. The mechanistic details reported in this study provide valuable insights into the catalytic activities of RsSTR in the synthesis of rare piperazinyl-indole skeletons. Furthermore, these findings are useful for guiding the rational design of STR enzymes with tailored capabilities for synthesizing novel alkaloid scaffolds.

Cytochrome P450 enzyme CYP11A1 is a key enzyme in the biosynthesis of pregnenolone; however, the detailed catalytic mechanism, including the underlying factors controlling regioselectivity in the highly sequential hydroxylation reactions, remains unclear. Here, we combined molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations to investigate the regioselective hydroxylation reactions catalyzed by CYP11A1. Our results indicate that the enzyme controls the hydroxylation site at different stages of the catalytic cycle depending on the substrate. For cholesterol (CH) hydroxylation, the regioselectivity is determined in the Cpd 0/substrate complex rather than in the Cpd I/substrate complex. Meanwhile, for the hydroxylation of the generated 22R-hydroxycholesterol (22 R -OHCH), the hydrogen-bonding interaction between the C22-OH of the substrate and the oxygen of Cpd I plays an important role in the selective hydroxylation of the C20 site and in promoting the adjustment of the substrate binding mode. In addition, a competing side reaction pathway for the hydroxylation of 22 R -OHCH is observed, in which the H-abstraction from the C22-OH group initiates C22-C20 bond cleavage and is followed by oxygen rebound. Overall, these results provide insights into the reaction mechanism of CYP11A1-catalyzed CH hydroxylation reactions and highlight the importance of the Cpd 0 species in understanding the catalytic selectivity of P450s and in the rational design of selective P450 enzymes.

Strictosidine synthase (STR) catalyzes in nature the enantioselective Pictet-Spengler condensation of secologanin and tryptamine to form (S)-strictosidine, a key tetrahydro-beta-carboline intermediate in the monoterpene indole alkaloid biosynthesis. Extensive studies have revealed that STR exhibits a broad substrate scope, being capable of accepting short-chain aliphatic and aromatic aldehydes and tryptamine derivatives with substitutions at different carbon positions. However, the activity toward N1-substituted tryptamine derivatives remained unexplored. To address this gap, in the present study, molecular dynamics simulations and quantum mechanical calculations were performed to identify the reasons responsible for the previously reported inability of STR in catalyzing the reaction of 1-methyltryptamine with secologanin. It was revealed that this inactivity originates from kinetically prohibitive catalytic steps, caused mainly by the steric clashes in the active site introduced by the N1-methyl group, rather than substrate binding limitations. Guided by the structural insights, short-chain aliphatic aldehydes were predicted to alleviate these steric constraints, supported by calculated feasible reaction barriers. Experimental validation confirmed this prediction, enabling the successful asymmetric synthesis of multiple N9-methyl-tetrahydro-beta-carboline derivatives. This work not only advances the fundamental understanding of STR catalysis but also establishes a combined computational-experimental strategy for exploring and extending the enzyme substrate scope.

2,5-Furandicarboxylic acid decarboxylase (HmfF) belongs to the UbiD family, which employs the cofactor prenylated flavin mononucleotide (prFMN) for catalysis. This enzyme catalyzes the reversible decarboxylation of 2,5-furandicarboxylic acid (FDCA) to produce 2-furancarboxylic acid (F2C). In the present study, quantum chemical calculations are employed to investigate the substrate binding mode and reaction mechanism of HmfF. The calculations demonstrate that HmfF follows a nucleophilic attack mechanism, rather than the 1,3-dipolar cycloaddition mechanism, which is believed more commonly adopted by the prFMN-dependent decarboxylases. Interestingly, the five-membered heterocyclic intermediate characteristic of 1,3-dipolar cycloaddition can also be located. However, it is only a fleeting intermediate that does not contribute to the catalysis. In the proposed mechanism, the reaction initiates with a single CC bond formation between FDCA and prFMN. Then, the CC bond between the carboxylate and the furan group of FDCA breaks to release CO 2 $\left(\text{CO}\right)_{2}$ , followed by a proton transfer from Glu259 to the decarboxylated intermediate, and the subsequent CC bond cleavage to generate the F2C product. Additionally, the infeasibility of HmfF in promoting the decarboxylation of F2C is evaluated computationally, and the obtained information is helpful in designing mutations to enable this reactivity.

Enzymatic carboxylation of phenols via the Kolbe-Schmitt reaction represents a promising sustainable strategy for $CO_2$ fixation and synthesis of high-value chemicals. This study investigates the reaction mechanism of 5-carboxyvanillate decarboxylase (LigW)-catalyzed carboxylation of non-natural monohydroxybenzoic acids, employing the quantum chemical cluster approach. First, we investigate the carboxylation mechanism of 4-hydroxybenzoate (4-HBA) to produce 4-hydroxyisophthalate (4-HIPA), a dicarboxylic acid with potent antioxidant and neuroprotective applications. The calculations reveal that the $CO_2$-binding mediates the preferred binding mode of the substrates and the incorporation of $CO_2$ to the active site favors the mode beneficial for the following reaction. The chemical reaction is initiated by the formation of a carbon-carbon bond between $CO_2$ and 4-HBA, followed by the rate-limiting proton transfer from the active site residue Asp314 to the resulting intermediate of the first step with a calculated barrier of 19.2 kcal/mol. Additionally, the potential of LigW in catalyzing the carboxylation of 3-hydroxybenzoate (3-HBA) is evaluated, and the calculations show that the reaction is energetically unfeasible due to the prohibitively high barrier of chemical steps. These mechanistic insights, together with the previous studies on the natural substrate, provide important information for the rotational design of LigW variants for industrial biocatalysis and $CO_2$ utilization.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the conversion of 5-enolpyruvate (PEP) and shikimic acid phosphate (S3P) to 5-enolpyruvylshikimic acid-3-phosphate (EPSP), releasing inorganic phosphate. This reaction is the sixth step of the shikimate pathway, which is a metabolic pathway used by microorganisms and plants for the biosynthesis of aromatic amino acids and folates but not in mammals. In the present study, the detailed reaction mechanism of EPSPS from Nicotiana tabacum (NtEPSPS) is revealed by quantum chemical calculations with the cluster approach. The reaction is proposed to involve the formation of a carbocation intermediate, the formation of a tetrahedral intermediate, the C-O bond cleavage and the re-formation of C=C bond. All four steps are concerted processes involving proton transfer events. The calculations suggest a step-wise mechanism for the formation of the tetrahedral intermediate by the proton transfer from the hydroxyl group of S3P to Asp331 and the nucleophilic attack of hydroxyl group on the carbocation, which is consistent with the proposal in literature. The energy profile for the entire reaction is presented, showing that C-O bond cleavage of the tetrahedral intermediate, releasing phosphate, is the rate-limiting step. The interaction between the Glu359 residue and the phosphate group is significant in stabilizing the phosphate.

Various metal-organic frameworks (MOFs) containing trivalent cations (such as Fe3+, Al3+, and Cr3+) have been reported and have shown great potential in applications. However, the high structural diversity and strong electronic interactions between metal centers and their ligands make the molecular dynamics simulations of MOFs challenging. In this work, we developed new dummy atom models for Fe3+, Al3+, and Cr3+ cations, which can be used in classical molecular dynamics simulations of MOFs. In our models, the correct solvation free energies and metal-ligand distances can be simultaneously reproduced. Furthermore, the usefulness and transferability of our models were validated using the commonly studied MIL-100(M) (M = Fe3+, Al3+, Cr3+) and MIL-88B(Fe3+) systems. Our developed models offer a valuable tool for simulating complex systems containing Fe3+, Al3+, and Cr3+ cations with octahedral coordination structures.

myo-Inositol, a water-soluble B vitamin compound, has broad applications in the food, pharmaceutical, and feed industries. Sustainable production of myo-inositol from starch can be achieved using an in vitro synthetic enzymatic biosystem (ivSEB) comprising four key enzymes. The NAD+/NADH self-recycling hyperthermophilic inositol 1-phosphate synthase (AfIPS) from Archaeoglobus fulgidus catalyzes the rate-limiting reaction. Utilizing a combinatorial active-site saturation test and iterative saturation mutagenesis (CAST/ISM), an optimized AfIPS mutant (I11C/I334V) was obtained. This mutant retained thermal stability comparable to the wild-type enzyme and exhibited a 2-fold increase in the specific activity (1.80 to 3.83 U/mg at 70 °C), and a 3-fold improvement in catalytic efficiency (kcat/Km: 7.46 to 22.1 mM-1 min-1). Molecular dynamics (MD) simulations revealed a novel hydrogen bond between the C11 side chain and the NAD+ pyrophosphate, enhancing cofactor binding and stabilizing the active conformation. This stabilization promotes optimal substrate alignment and improved hydride transfer, reducing total enzyme loading in the ivSEB by approximately 40% at varying substrate levels. These findings highlight the potential of semirational engineering of rate-limiting enzymes to enhance process efficiency and reduce costs, thus advancing the feasibility of scalable and economically sustainable myo-inositol production.

Forazoline A, produced by the marine actinomycete Actinomadura sp. WMMB-499, is a unique PK/NRP hybrid macrolactone with promising antifungal in vivo efficacy through a previously unreported mechanism. Although a PKS/NRPS gene cluster was identified as a candidate for forazoline production, the precise biosynthetic pathway and the functions of the tailoring enzymes remain unclear. In this work, the functions of three cytochrome P450 mono-oxygenases (FrazP1P2P3) were characterized. Notably, FrazP2 was found to mediate cyclohexane ring formation from an 1,3,6-triene precursor during forazoline A biosynthesis, as confirmed by genetic and biochemical analysis. To gain structural and mechanistic insight into the activity of FrazP2, the crystal structure of a FrazP2-substrate complex has been solved at 2.3 Å resolution. The molecular dynamics simulations and DFT calculations revealed an unprecedented enzyme-catalyzed oxidative cyclization reaction by FrazP2. These findings expand our understanding of the catalytic diversity of cytochrome P450s, contributing to the diversification of natural products and enabling the creation of unnatural derivatives with increased antifungal potency.

The enzymatic atroposelective synthesis of biaryl compounds is relatively rare, despite considerable attention received by biocatalysis in the academic and industrial sectors. Imine reductases (IREDs) are an important class of enzymes that have been applied in the asymmetric synthesis of chiral amine building blocks. In this study, two IREDs (IR140 and IR189) were identified to catalyze the efficient desymmetrization of biaryls utilizing various amine donors. Further protein engineering enabled the identification of variants (IR189 M8–M9 and IR189 M13–M14) that are able to catalyze the formation of both ( R ) and ( S ) atropisomers in excellent yields and atroposelectivities (24 examples, up to 99 % ee and yield). The absolute configuration and rotational barriers were confirmed, and the reactions were readily scaled up to allow isolation of the atropisomeric product in 99 % ee and 82 % yield. The optically pure biaryl amines were further derivatized into various synthetically useful atropisomers. To shed light on the molecular recognition mechanisms, molecular dynamics (MD) simulations were performed, offering plausible explanations for the improved atroposelectivity and enzymatic activity. The current strategy expands the scope of the IRED-catalyzed synthesis of axially chiral biaryl amines, contributing significantly to the field of atroposelective biocatalysis.

Electron transport through metal-organic frameworks by a hopping mechanism between discrete redox active sites is coupled to diffusion-migration of charge-balancing counter cations. Experimentally determined apparent diffusion coefficients, Deapp, that characterize this form of charge transport thus contain contributions from both processes. While this is well established for MOFs, microscopic descriptions of this process are largely lacking. Herein, we systematically lay out different scenarios for cation-coupled electron transfer processes that are at the heart of charge diffusion through MOFs. Through systematic variations of solvents and electrolyte cations, it is shown that the Deapp for charge migration through a PIZOF-type MOF, Zr(dcphOH-NDI) that is composed of redox-active naphthalenediimide (NDI) linkers, spans over 2 orders of magnitude. More importantly, however, the microscopic mechanisms for cation-coupled electron propagation are contingent on differing factors depending on the size of the cation and its propensity to engage in ion pairs with reduced linkers, either non-specifically or in defined structural arrangements. Based on computations and in agreement with experimental results, we show that ion pairing generally has an adverse effect on cation transport, thereby slowing down charge transport. In Zr(dcphOH-NDI), however, specific cation-linker interactions can open pathways for concerted cation-coupled electron transfer processes that can outcompete limitations from reduced cation flux.

Salicylic acid decarboxylase (SDC) from the amidohydrolase superfamily (AHS) catalyzes the reversible decarboxylation of salicylic acid to form phenol. In this study, the substrate binding mode and reaction mechanism of SDC were investigated using computational and crystallographic methods. Quantum chemical calculations show that the enzyme follows the general mechanism of AHS decarboxylases. Namely, the reaction begins with proton transfer from a metal-coordinated aspartic acid residue (Asp298 in SDC) to the C1 of salicylic acid, which is followed by the C–C bond cleavage, to generate the phenol product and release CO2. Interestingly, the calculations show that SDC is a Mg-dependent enzyme rather than the previously proposed Zn-dependent, and the substrate is shown to be bidentately coordinated to the metal center in the catalysis, which is also different from the previous proposal. These predictions are corroborated by the crystal structure of SDC solved in complex with the substrate analogue 2-nitrophenol. The mechanistic insights into SDC in the present study provide important information for the rational design of the enzyme.

The control of site selectivity in asymmetric mono-hydrogenation of dienes or polyenes remains largely underdeveloped. Herein, we present a highly efficient desymmetrization of 1,4-dienes via iridium-catalyzed site- and enantioselective hydrogenation. This methodology demonstrates the first iridium-catalyzed hydrogenative desymmetriation of meso dienes and provides a concise approach to the installation of two vicinal stereogenic centers adjacent to an alkene. High isolated yields (up to 96 %) and excellent diastereo- and enantioselectivities (up to 99:1 d.r. and 99 % ee) were obtained for a series of divinyl carbinol and divinyl carbinamide substrates. DFT calculations reveal that an interaction between the hydroxy oxygen and the reacting hydride is responsible for the stereoselectivity of the desymmetrization of the divinyl carbinol. Based on the calculated energy profiles, a model that simulates product distribution over time was applied to show an intuitive kinetics of this process. The usefulness of the methodology was demonstrated by the synthesis of the key intermediates of natural products zaragozic acid A and (+)-invictolide.

Recently, the practical applications of tetravalent metal-based metal-organic frameworks (MOFs) have been drawing increasing attention due to their excellent stability and varied functionality. Understanding the behavior of MOFs at the atomic level is important for their rational design and postsynthetic optimization. In classical simulations, the cationic dummy atom model, where the charge of the metal ion is distributed to surrounding dummy particles, has shown robustness in providing the appropriate coordination geometry while retaining the flexibility in the metal ligand interaction. Here, we present a set of eight-coordinated tetravalent metal (Zr4+, Hf4+) dummy model force fields with 12-6-4 type Lennard-Jones potential for describing ion-induced dipole interactions. This model can reproduce both the experimental solvation free energy of the bare ion and good metal-ligand distances and has been validated as useful and transferable among MOFs with the same metal-core topology. The model is inherently modular since nodes and linkers are independently parametrized. It was tested with three different solvents which all led to stable structures. It also was been further validated with different metal node structures and different linkers, Zr/Hf mixed MOFs, and defects containing MOFs. Since the model is nonbonded, the process of metal-ligand substitutions could be studied. The flexibility of the model makes its applicability to chemical problems broad.

We present a highly efficient convergent asymmetric hydrogenation of E/Z mixtures of enamides catalyzed by N,P-iridium complexes supported by mechanistic studies. It was found that reduction of the olefinic isomers (E and Z geometries) produces chiral amides with the same absolute configuration (enantioconvergent hydrogenation). This allowed the hydrogenation of a wide range of E/Z mixtures of trisubstituted enamides with excellent enantioselectivity (up to 99% ee). A detailed mechanistic study using deuterium labeling and kinetic experiments revealed two different pathways for the observed enantioconvergence. For α-aryl enamides, fast isomerization of the double bond takes place, and the overall process results in kinetic resolution of the two isomers. For α-alkyl enamides, no double bond isomerization is detected, and competition experiments suggested that substrate chelation is responsible for the enantioconvergent stereochemical outcome. DFT calculations were performed to predict the correct absolute configuration of the products and strengthen the proposed mechanism of the iridium-catalyzed isomerization pathway.

We explore here a long-standing mechanistic question by using quantum-mechanical/molecular-mechanical (QM/MM) methodology. The question concerns the mechanism of steroid hormone biosynthesis, whereby the P450 enzyme, CYP11A1, catalyzes the C20-C22 bond-cleavage in the 20,22-hydroxylated cholesterol, 20R,22R-DiOHCH, leading to pregnenolone, which is critical for the subsequent production of all steroid hormones. This is an unusual feat whereby the P450 enzyme breaks two O-H bonds and one C-C bond, while making two C=O bonds. How does the enzyme perform such a complex and highly energy-demanding reaction? Our computational results rule out the previously proposed Compound I (Cpd I) electrophilic attack mechanism via the formation of a peroxide intermediate as well as the H-abstraction-mediated C-C cleavage mechanism. Notably, oxygen-rebound cannot transpire, in spite of the fact that the classical active species, Cpd I, participates in the catalytic process. Our findings reveal a mechanism whereby C-C bond cleavage is mediated by an electron transfer from the C22-O--deprotonated substrate to Cpd I. As such, our QM/MM calculations demonstrate that Cpd I acts as an electron sink that facilitates the C-C bond cleavage.

Solanum nigrum fruits have been conventionally used in beverages due to their nutritional substances such as minerals, vitamins, amino acids, proteins, sugars, polyphenols, and anthocyanins. The characterization of components and regulatory mechanism of anthocyanins in S. nigrum fruits have rarely been reported. In this study, we determined that the peel and flesh of S. nigrum fruits shared similar HPLC profiles but different contents and total antioxidant activities for anthocyanins. After an efficient purification method, mainly including extraction with pH 1.0 distilled water and then desorption with pH 1.0 95% ethanol after a DM-130 resin adsorption step to obtain more pure anthocyanin extracts, the purity of anthocyanins extracted from S. nigrum fruits reached 56.1%. Moreover, eight anthocyanins from S. nigrum fruit were identified with HPLC-MS/MS for the first time. A typical R2R3-MYB transcription factor gene, SnMYB, was also cloned for the first time by rapid amplification of cDNA ends (RACE)-PCR from S. nigrum. Moreover, the contents of anthocyanins were shown to correlate well (r = 0.93) with the expression levels of SnMYB gene during the fruit's developmental stages. Most significantly, SnMYB gene successfully produced high anthocyanin content (1.03 mg/g) when SnMYB gene was transiently expressed in tobacco leaves. Taken together, S. nigrum fruits are a promising resource for anthocyanin extraction, and SnMYB gene is an activator that positively regulates anthocyanin biosynthesis in S. nigrum.