A conserved mechanism affecting hydride shifting and deprotonation in the synthesis of hopane triterpenes as compositions of wax in oat.

Triterpenoids are biologically active metabolites synthesized from a common linear precursor catalyzed by 2,3-oxidosqualene cyclases (OSCs) to form diverse triterpenoid skeletons. OSCs corresponding to many discovered triterpene alcohols in nature have not been functionally and mechanistically characterized due to the diversity of chemical structures and complexity of the cyclization mechanism. We carried out a genome-wide investigation of OSCs from Avena strigosa and discovered two triterpene synthases, namely, AsHS1 and AsHS2, using a Nicotiana benthamiana expression system. These synthases produce hopenol B and hop-17(21)-en-3ß-ol, which are components of surface wax in oat panicles and sheathes, respectively. We demonstrated that substitutions of two to three amino acid residues in AsHS1 with corresponding residues from AsHS2 allowed it to be completely converted into a hop-17(21)-en-3ß-ol synthase. AsHS2 mutants with a substitution at site 410 could synthesize hopenol B alone or mixed with a side product isomotiol. The combined quantum mechanics and molecular mechanics calculation demonstrated that the side chain size of the residue at site 410 regulated the relative orientations between the hopyl C22 cation and Phe257, leading to a difference in deprotonation positions through providing or not providing cation­π interaction between the aromatic ring of F257 and the carbocation intermediate. A similar mechanism could be applied to a hopenol B synthase from a dicotyledonous plant Aquilegia. This study provided mechanistic insight into triterpenoid synthesis and discovered key amino acid residues acting on hydride transfer and a deprotonation site to differentiate between hopane-type scaffolds in diverse plant species.

Bio-inspired chemical space exploration of terpenoids.

Many computational methods are devoted to rapidly generating pseudo-natural products to expand the open-ended border of chemical spaces for natural products. However, the accessibility and chemical interpretation were often ignored or underestimated in conventional library/fragment-based or rule-based strategies, thus hampering experimental synthesis. Herein, a bio-inspired strategy (named TeroGen) is developed to mimic the two key biosynthetic stages (cyclization and decoration) of terpenoid natural products, by utilizing physically based simulations and deep learning models, respectively. The precision and efficiency are validated for different categories of terpenoids, and in practice, more than 30 000 sesterterpenoids (10 times as many as the known sesterterpenoids) are predicted to be linked in a reaction network, and their synthetic accessibility and chemical interpretation are estimated by thermodynamics and kinetics. Since it could not only greatly expand the chemical space of terpenoids but also numerate plausible biosynthetic routes, TeroGen is promising for accelerating heterologous biosynthesis, bio-mimic and chemical synthesis of complicated terpenoids and derivatives.

NIMO: A Natural Product-Inspired Molecular Generative Model Based on Conditional Transformer.

Natural products (NPs) have diverse biological activity and significant medicinal value. The structural diversity of NPs is the mainstay of drug discovery. Expanding the chemical space of NPs is an urgent need. Inspired by the concept of fragment-assembled pseudo-natural products, we developed a computational tool called NIMO, which is based on the transformer neural network model. NIMO employs two tailor-made motif extraction methods to map a molecular graph into a semantic motif sequence. All these generated motif sequences are used to train our molecular generative models. Various NIMO models were trained under different task scenarios by recognizing syntactic patterns and structure-property relationships. We further explored the performance of NIMO in structure-guided, activity-oriented, and pocket-based molecule generation tasks. Our results show that NIMO had excellent performance for molecule generation from scratch and structure optimization from a scaffold.

Structural Insights into Three Sesquiterpene Synthases for the Biosynthesis of Tricyclic Sesquiterpenes and Chemical Space Expansion by Structure-Based Mutagenesis.

The cyclization of farnesyl diphosphate (FPP) into highly strained polycyclic sesquiterpenes is challenging. We here determined the crystal structures of three sesquiterpene synthases (STSs, namely, BcBOT2, DbPROS, and CLM1) catalyzing the biosynthesis of the tricyclic sesquiterpenes presilphiperfolan-8ß-ol (1), Δ6-protoilludene (2), and longiborneol (3). All three STS structures contain a substrate mimic, the benzyltriethylammonium cation (BTAC), in their active sites, providing ideal templates for quantum mechanics/molecular mechanics (QM/MM) analyses toward their catalytic mechanisms. The QM/MM-based molecular dynamics (MD) simulations revealed the cascade reactions toward the enzyme products, and different key active site residues that play important roles in stabilizing reactive carbocation intermediates along the three pathways. Site-directed mutagenesis experiments confirmed the roles of these key residues and concomitantly resulted in 17 shunt products (4-20). Isotopic labeling experiments addressed the key hydride and methyl migrations toward the main and several shunt products. These combined methods provided deep insights into the catalytic mechanisms of the three STSs and demonstrated how the chemical space of STSs can rationally be expanded, which may facilitate applications in synthetic biology approaches toward pharmaceutical and perfumery agents.

QM/MM Modeling Aided Enzyme Engineering in Natural Products Biosynthesis.

Natural products and their derivatives are widely used across various industries, particularly pharmaceuticals. Modern engineered biosynthesis provides an alternative way of producing and meeting the growing need for diverse natural products. Natural enzymes, on the other hand, often exhibit unsatisfactory catalytic characteristics and necessitate further enzyme engineering modifications. QM/MM, as a powerful and extensively used computational tool in the field of enzyme catalysis, has been increasingly applied in rational enzyme engineering over the past decade. In this review, we summarize recent advances in QM/MM computational investigation on enzyme catalysis and enzyme engineering for natural product biosynthesis. The challenges and perspectives for future QM/MM applications aided enzyme engineering in natural product biosynthesis will also be discussed.

A DFT study of the endo-selectivity mechanism of the Diels-Alder reaction in lindenane dimeric sesquiterpene synthesis promoted by pyridines.

The lindenane dimeric sesquiterpenoids with versatile biological activities are accessible via biometric synthesis, in which the endo-selective Diels-Alder reaction plays an important role. To explore the endo-selectivity of the Diels-Alder reaction between lindenane sesquiterpenes promoted by pyridines, density functional theory (DFT) calculations were performed to explore the reaction mechanism between pyridines and D-A monomers. The calculations performed on the reaction pathways explain why pyridines can promote endo-selectivity via hydrogen bonding, and the hydrogen bond strength is a key factor driving the Diels-Alder reaction in major biochemical systems. These DFT-level insights will pave the way for designing better promoters for Diels-Alder reactions in biometric synthesis applications.

Infection Characteristics and Drug Susceptibility of Multidrug-Resistant Bacteria in Patients with Diabetic Foot Ulcers.

BACKGROUND: We aimed to analyze the infection characteristics of multidrug-resistant organisms (MDROs) and their resistance to antibiotics in patients with diabetic foot and provide guidance for the use of antibiotics in clinical practice. METHODS: The clinical data of 737 patients with diabetic foot who were hospitalized at our institution from February 2020 to January 2023 were retrospectively analyzed. Purulent secretions were collected from the patient's ulcers and bacterial culture, identification, and drug susceptibility tests were performed. The multidrug resistance (MDR) rate of different bacteria, composition ratio of MDROs, drug resistance characteristics of the main MDROs, distribution characteristics of multidrug-resistant gram-positive cocci and gram-negative bacilli in patients with different Wagner Grades, MDR in patients with different Wagner Grades, bacterial infection rate, and other indicators were analyzed. RESULTS: Pathogenic bacteria from wound secretions of 505 patients were cultured, and 509 pathogenic bacteria were obtained. Among the pathogenic bacteria, 225 strains were gram-positive cocci, of which 172 (76.44%) were MDROs, and 284 were gram-negative bacilli, of which 232 (81.69%) were MDROs. Among the 404 multidrug-resistant strains, gram-positive cocci and gram-negative bacilli accounted for 42.57% and 57.43%, respectively. The top five dominant MDROs were Staphylococcus aureus (18.56%), coagulase-negative Staphylococcus (10.89%), Escherichia coli (10.15%), Proteus mirabilis (8.17%), Proteus vulgaris (6.19%), and Pseudomonas aeruginosa (6.19%). Staphylococcus aureus and coagulase-negative Staphylococcus were more resistant to penicillin, oxacillin, erythromycin, azithromycin, and clarithromycin, with resistance rates of 50.0 - 95.0%. The resistance rates of E. coli to ampicillin, cefazolin, cefuroxime, ceftriaxone, and cefepime were > 75%. With an increase in Wagner Grade, the proportion of gram-negative bacilli among the pathogenic bacteria of MDROs increased significantly (p < 0.05), as did the infection rate of MDROs in patients with diabetic foot (χ2 = 14.045, p < 0.05). CONCLUSIONS: MDROs in patients with diabetic foot are mainly gram-negative bacilli, followed by gram-positive cocci. The drug resistance of various MDROs varies greatly. With the increase in Wagner Grade and MDR of diabetic foot patients, the infection rate of drug-resistant bacteria has increased significantly. Therefore, clinicians should use drugs rationally according to drug sensitivity results.

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC.

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and ß-amyrin, respectively.

Chemotaxonomic investigation of plant terpenoids with an established database (TeroMOL).

Terpenoids constitute the biggest class of plant-derived natural products with diverse chemical structures and extensive biological activities. Interpreting enzyme functions and mining new structures of terpenoids could be inspired by the cheminformatic and chemotaxonomic analysis, whereas it is hampered by the incompleteness of available data for terpenoids. Here a dedicated terpenoids database, TeroMOL, is developed to collect more than 170 000 terpenoids and their derivatives annotated with reported biological sources, along with a user-friendly and freely accessible webserver to visualise and analyse the terpenoids skeletons and organism sources. The quantitative distributions as well as the qualitative trends between terpenoid skeletons and organism sources in plant kingdom are revealed from a chemotaxonomic view, while no comparisons are attempted due to the inherent data biases. Nevertheless, the terpenoid chemomarkers in several organisms are discussed based on the available data with highly enriched and exclusive carbon skeletons. We believe that the TeroMOL database and its accessory computational tools will be very promising for exploring the chemical space and biological sources of terpenoids, and assisting the terpenoid research community in the future.

GM-DockZn: a geometry matching-based docking algorithm for zinc proteins.

MOTIVATION: Molecular docking is a widely used technique for large-scale virtual screening of the interactions between small-molecule ligands and their target proteins. However, docking methods often perform poorly for metalloproteins due to additional complexity from the three-way interactions among amino-acid residues, metal ions and ligands. This is a significant problem because zinc proteins alone comprise about 10% of all available protein structures in the protein databank. Here, we developed GM-DockZn that is dedicated for ligand docking to zinc proteins. Unlike the existing docking methods developed specifically for zinc proteins, GM-DockZn samples ligand conformations directly using a geometric grid around the ideal zinc-coordination positions of seven discovered coordination motifs, which were found from the survey of known zinc proteins complexed with a single ligand. RESULTS: GM-DockZn has the best performance in sampling near-native poses with correct coordination atoms and numbers within the top 50 and top 10 predictions when compared to several state-of-the-art techniques. This is true not only for a non-redundant dataset of zinc proteins but also for a homolog set of different ligand and zinc-coordination systems for the same zinc proteins. Similar superior performance of GM-DockZn for near-native-pose sampling was also observed for docking to apo-structures and cross-docking between different ligand complex structures of the same protein. The highest success rate for sampling nearest near-native poses within top 5 and top 1 was achieved by combining GM-DockZn for conformational sampling with GOLD for ranking. The proposed geometry-based sampling technique will be useful for ligand docking to other metalloproteins. AVAILABILITY AND IMPLEMENTATION: GM-DockZn is freely available at www.qmclab.com/ for academic users. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

AromTool: predicting aromatic stacking energy using an atomic neural network model.

Aromatic stacking exists widely and plays important roles in protein-ligand interactions. Computational tools to automatically analyze the geometry and accurately calculate the energy of stacking interactions are desired for structure-based drug design. Herein, we employed a Behler-Parrinello neural network (BPNN) to build predictive models for aromatic stacking interactions and further integrated it into an open-source Python package named AromTool for benzene-containing aromatic stacking analysis. Based on extensive testing, AromTool presents desirable precision in comparison to DFT calculations and excellent efficiency for high-throughput aromatic stacking analysis of protein-ligand complexes.

Residue-Orientation-Dependent Dynamics and Selectivity of Active Pocket in Microbe Class I Terpene Cyclases.

Microbe class I terpene cyclases (TPCs) are responsible for deriving numerous functionally and structurally diverse groups of terpenoid natural products. The conformational change of their active pockets from "open" state to "closed" state upon substrate binding has been clarified. However, the key structural basis relevant to this active pocket dynamics and its detailed molecular mechanism are still unclear. In this work, on the basis of the molecular dynamics (MD) on two microbe class I TPCs (SdS and bCinS), we propose that the active pocket dynamics is highly dependent on the residue orientation of two conserved structural bases R-D dyad and X-R-D triad, rather than the previously suggested flexibility of kink region. Actually, we considered that the flexibility of kink region is synchronous with the R residue orientation of the X-R-D triad, which could regulate the entrance size of active pocket and thus affect the substrate selectivity of active pocket by utilizing the promiscuity of the X-R-D triad. Furthermore, to better understand the function of the two structural bases, two intelligible models of "PPi catcher-locker" and "selector-PPi sensor-orienter" are proposed to, respectively, describe the R-D dyad and X-R-D triad and broadened to more microbe class I TPCs. These findings exhibit the dynamics of active pocket inaccessible in static crystal structures and provide useful structural basis knowledge for further design of microbe class I TPCs with different cyclization ability.

TeroKit: A Database-Driven Web Server for Terpenome Research.

Natural products are the major resource of drug discovery, and terpenoids represent the largest family of natural products. Terpenome is defined as all terpenoid-like and terpenoid-derived natural compounds, including the terpenoids, steroids, and their derivatives. Herein, aiming to navigate the chemical and biological space of terpenome, the first comprehensive database dedicated to terpenome research has been developed by collecting over 110 000 terpenome molecules from various resources, distributed in 14 351 species, belonging to 1109 families, and showing activity against 1366 biological targets. Much of the publically available information or computationally predicted properties for each terpenome molecule is annotated and integrated into TeroKit (http://terokit.qmclab.com/), serving as free Web server for academic use. Moreover, several practical toolkits, such as target profiling and conformer generation modules, are also implemented to facilitate the drug discovery of terpenome.

Recognition of PDL1/L2 by different induced-fit mechanisms of PD1: a comparative study of molecular dynamics simulations.

Recent clinical data has shown that some cancers choose to express PDL2 compared to PDL1. Therefore, a detailed and comparative study of the dynamic binding mechanism between PD1/PDL1 and PD1/PDL2 can guide drug design towards PD1. Herein, long time-scale classical molecular dynamics simulation, binding free energy calculation, energy decomposition and homology modeling for PD1/PDL2 were used to shed light on the differences in the binding mechanisms of the PD1/PDL1 and PD1/PDL2 complexes. On one hand, our results reveal a different binding mechanism of PD1 binding to PDL1 and PDL2, which is mainly attributed to the induced-fit from different proteins, that is, the C'D loop of PD1 is essential for PD1/PDL1, while the CD loop of PDL2 is critical for PD1/PDL2. Particularly, the "enclosed" conformation of PDL2 leads to a higher affinity between PD1-PDL2 in comparison to the affinity between PD1-PDL1. For PD1/PDL1, the key residues of N66, Y68, Q75, T76, K78, D85, I126, L128, A132, I134 and E136 are the dominant residues for stabilizing the protein-protein interaction (PPI). For PD1/PDL2, the key residues are mainly concentrated in the FG loop, including N33/Q75/L128/A132/Q133/I134/K135. These findings provide a comprehensive understanding of the distinctive binding kinetics and thermodynamic features, which will contribute meaningfully for the design of peptides and small molecule inhibitors to selectively break the PPI interfaces of PD1/PDL1 and PD1/PDL2.

Asymmetric Counter-Anion-Directed Aminomethylation: Synthesis of Chiral ß-Amino Acids via Trapping of an Enol Intermediate.

A novel enantioselective aminomethylation reaction of diazo compound, alcohol and α-aminomethyl ether enabled by asymmetric counteranion-directed catalysis is disclosed that offers an efficient and convenient access to furnish optically active α-hydroxyl-ß-amino acids in high yield with high to excellent enantioselectivities. Control experiments and DFT calculations indicate that the transformation proceeds through trapping the in situ generated enol intermediate with methylene iminium ion, and the asymmetric induction was enabled by chiral pentacarboxycyclopentadiene anion via H-bonding and electrostatic interaction.

Development of a Smart Fluorescent Sensor That Specifically Recognizes the c-MYC G-Quadruplex.

The specific sensing of an exact G-quadruplex structure by small molecules has never been reported. A fluorescent sensor based on the photoinduced electron transfer (PeT) mechanism provides possibilities for such specific, one-to-one recognition, indicated by fluorescence. We have rationally developed a PeT fluorescent sensor IZFL-2 by linking triarylimidazole and fluorescein moieties. IZFL-2 is a distinctive, smart sensor whose fluorescence is tunable by its molecular conformations. We then applied IZFL-2 to sensing G-quadruplexes and found that it could exactly distinguish the wild-type c-MYC G-quadruplex from other types of G-quadruplexes, as shown by the activation of its fluorescence. To understand this behavior, we performed various experiments, including fluorescence assays, absorption assays, and multiscale molecular dynamics simulations, to thoroughly investigate the optimal binding mode of IZFL-2 in the c-MYC G-quadruplex. Then, the corresponding HOMO-LUMO of IZFL-2 was analyzed, and the results demonstrated that the PeT process of IZFL-2 is suppressed only in the wild-type c-MYC G-quadruplex via specific loop interactions, which restores its fluorescence. To our knowledge, this smart molecule provides the first example of and new insights into the development of sensors specific for a particular G-quadruplex structure by utilizing intramolecular PeT-controlled fluorescence switching.

Exploring Chemical and Biological Space of Terpenoids.

Terpenoids represent the largest family of natural products (NPs) with dramatically chemical and structural diversity, which makes terpenoids the important compound resources of drug discovery. However, comprehensive understanding on the structure-function features for terpenoid NPs is limited. In this work, we have systematically explored the chemical and biological space of terpenoid NPs, including their distribution, physicochemical properties, scaffold features, and functional applications, by utilizing various cheminformatics and bioinformatics approaches. We have not only confirmed that terpenoid NPs have good drug-likeness and great potential for drug discovery but, more importantly, illuminated the uniqueness of cyclic scaffold diversity in different species (plants, fungi, bacteria, and animals) and the specificity of biological function for the dominant fused-ring scaffolds of terpenoids. The present work supplies a valuable reference for identifying the new structure and unknown function of terpenoid NPs.

Conformational dynamics and allosteric effect modulated by the unique zinc-binding motif in class IIa HDACs.

Class IIa histone deacetylases (HDACs) have been considered as potential targets for the treatment of several diseases. Compared to other HDACs, class IIa HDACs have an additional second zinc binding motif. So far, the function of the unique zinc-binding motif is still not very clear. In this work, extensive classical molecular dynamics (MD) simulations were employed to illuminate the conformational change modulated by the unique zinc-binding motif. It has been revealed that the unique zinc-binding motif is a crucial structural stabilization factor in retaining the catalytic activity of the enzyme and the stability of the multi-protein complex, by remotely modulating the active site pocket in a "closed" conformation. Moreover, it is also revealed that the Loop2 motion in HDAC4 is less flexible than that in HDAC7, which opens a new avenue to design selective inhibitors by utilizing the local conformational dynamics difference between the structurally highly similar HDAC4 and HDAC7. Finally, by comparative studies with class I HDACs (HDAC1-3), it is found that the reversible "in-out" conformational transformation of the binding rail (highly conserved both in class I and IIa HDACs) occurs spontaneously in HDAC1-3, whereas the binding rail is trapped in an "in" conformation owing to the strong metal coordination interaction of the unique CCHC zinc-binding motif in class IIa HDACs. Thus, the CCHC zinc-binding motif may be a feasible allosteric site for the development of class IIa-selective inhibitors.

Catalytic promiscuity of the non-native FPP substrate in the TEAS enzyme: non-negligible flexibility of the carbocation intermediate.

The TEAS, one of the sesquiterpene cyclases (FPPC), shows enzyme promiscuity that can effectively catalyze both the natural substrate (trans,trans)-FPP and the non-native (cis,trans)-FPP substrate to generate diverse products/byproducts. So far, the catalytic mechanism of the promiscuous substrate is still unclear. In this work, QM(DFT)/MM MD simulations were employed to illuminate the predominant 1,6-closure pathway reaction mechanism for the non-native substrate (cis,trans)-FPP, while the 1,10-closure pathway is the major reaction for the native substrate. It has been revealed that the catalytic promiscuity of TEAS is mostly attributable to the notable conformational dynamics of the branching intermediate bisabolyl cation. The comparative studies to FSTS (another widely studied FPPC) further indicate that the intrinsic intermediate flexibility in TEAS is highly correlated to the plasticity of the enzyme active site pocket contour. Finally, we propose a general picture for controlling the promiscuity and fidelity in FPPC catalysis, including substrate folding, intermediate flexibility and key residues.

Intrinsic Dynamics of the Binding Rail and Its Allosteric Effect in the Class I Histone Deacetylases.

The development of novel isoform/class-selective inhibitors is still of great biological and medical significance to conquer the continuously reported side effects for the histone deacetylase (HDAC) drugs. The first potent HDAC allosteric inhibitor was discovered last year, and this allosteric inhibitor design is thought to be a promising strategy to overcome the current challenges in HDAC inhibitor design. However, the detailed allosteric mechanism and its remote regulatory effects on the catalytic/inhibitor activity of HDAC are still unclear. In this work, on the basis of microsecond-time-scale all-atom molecular dynamics (MD) simulations and picosecond-time-scale density functional theory/molecular mechanics MD simulations on HDAC8, we propose that the allostery is achieved by the intrinsic conformational flexibility of the binding rail (constituted by a highly conserved X-D residue dyad), which steers the loop-loop motion and creates the diverse shapes of the allosteric sites in different HDAC isoforms. Additionally, the rotatability of the binding rail is an inherent structural feature that regulates the hydrophobicity of the linker binding channel and thus further affects the HDAC enzyme inhibitory/catalytic activity by utilizing the promiscuity of X-D dyad. Since the plastic X residue is different among class I HDACs, these new findings provide a deeper understanding of the allostery, which is guidable for the design of new allosteric inhibitors toward the allosteric site and structure modifications on the conventional inhibitors binding into the active pocket by exploiting the intrinsic dynamic features of the conserved X-D dyad.