Enhanced Sampling Molecular Dynamics Simulations Reveal Transport Mechanism of Glycoconjugate Drugs through GLUT1.

Glucose transporters GLUT1 belong to the major facilitator superfamily and are essential to human glucose uptake. The overexpression of GLUT1 in tumor cells designates it as a pivotal target for glycoconjugate anticancer drugs. However, the interaction mechanism of glycoconjugate drugs with GLUT1 remains largely unknown. Here, we employed all-atom molecular dynamics simulations, coupled to steered and umbrella sampling techniques, to examine the thermodynamics governing the transport of glucose and two glycoconjugate drugs (i.e., 6-D-glucose-conjugated methane sulfonate and 6-D-glucose chlorambucil) by GLUT1. We characterized the specific interactions between GLUT1 and substrates at different transport stages, including substrate recognition, transport, and releasing, and identified the key residues involved in these procedures. Importantly, our results described, for the first time, the free energy profiles of GLUT1-transporting glycoconjugate drugs, and demonstrated that H160 and W388 served as important gates to regulate their transport via GLUT1. These findings provide novel atomic-scale insights for understanding the transport mechanism of GLUT1, facilitating the discovery and rational design of GLUT1-targeted anticancer drugs.

Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Engineering the Relatively Conserved Residues in Active Site Architecture of Thermophilic Chitinase SsChi18A Enhanced Catalytic Activity.

Chitinase plays a vital role in the efficient biotransformation of the chitin substrate. This study aimed to modify and elucidate the contribution of the relatively conserved residues in the active site architecture of a thermophilic chitinase SsChi18A from Streptomyces sp. F-3 in processive catalysis. The enzymatic activity on colloidal chitin increased to 151%, 135%, and 129% in variants Y286W, E287A, and K186A compared with the wild type (WT). Also, the apparent processive parameter G2/G1 was lower in the variants compared to the WT, indicating the essential role of Tyr-286, Glu-287, and Lys-186 in processive catalysis. Additionally, the enzymatic activity on the crystalline chitin of F48W and double mutants F48W/Y209F and F48W/Y286W increased by 35%, 16%, and 36% compared with that for WT. Molecular dynamics simulations revealed that the driving force of processive catalysis might be related to the changes in interaction energy. This study provided a rational design strategy targeting relatively conserved residues to enhance the catalytic activity of GH18 processive chitinases.

Structure-Interaction Relationship of Polymyxins with Lung Surfactant.

Multidrug-resistant Gram-negative bacteria present an urgent and formidable threat to the global public health. Polymyxins have emerged as a last-resort therapy against these 'superbugs'; however, their efficacy against pulmonary infection is poor. In this study, we integrated chemical biology and molecular dynamics simulations to examine how the alveolar lung surfactant significantly reduces polymyxin antibacterial activity. We discovered that lung surfactant is a phospholipid-based permeability barrier against polymyxins, compromising their efficacy against target bacteria. Next, we unraveled the structure-interaction relationship between polymyxins and lung surfactant, elucidating the thermodynamics that govern the penetration of polymyxins through this critical surfactant layer. Moreover, we developed a novel analog, FADDI-235, which exhibited potent activity against Gram-negative bacteria, both in the presence and absence of lung surfactant. These findings shed new light on the sequestration mechanism of lung surfactant on polymyxins and importantly pave the way for the rational design of new-generation lipopeptide antibiotics to effectively treat Gram-negative bacterial pneumonia.

Loop Evolutionary Patterns Shape Catalytic Efficiency of TRI101/201 for Trichothecenes: Insights into Protein-Substrate Interactions.

Trichothecenes are highly toxic mycotoxins produced by Fusarium fungi, while TRI101/201 family enzymes play a crucial role in detoxification through acetylation. Studies on the substrate specificity and catalytic kinetics of TRI101/201 have revealed distinct kinetic characteristics, with significant differences observed in catalytic efficiency toward deoxynivalenol, while the catalytic efficiency for T-2 toxin remains relatively consistent. In this study, we used structural bioinformatics analysis and a molecular dynamics simulation workflow to investigate the mechanism underlying the differential catalytic activity of TRI101/201. The findings revealed that the binding stability between trichothecenes and TRI101/201 hinges primarily on a hydrophobic cage structure within the binding site. An intrinsic disordered loop, termed loop cover, defined the evolutionary patterns of the TRI101/201 protein family that are categorized into four subfamilies (V1/V2/V3/M). Furthermore, the unique loop displayed different conformations among these subfamilies' structures, which served to disrupt (V1/V2/V3) or reinforce (M) the hydrophobic cages. The disrupted cages enhanced the water exposure of the hydrophilic moieties of substrates like deoxynivalenol and thereby hindered their binding to the catalytic sites of V-type enzymes. In contrast, this water exposure does not affect substrates like T-2 toxin, which have more hydrophobic substituents, resulting in a comparable catalytic efficiency of both V- and M-type enzymes. Overall, our studies provide theoretical support for understanding the catalytic mechanism of TRI101/201, which shows how an intrinsic disordered loop could impact the protein-ligand binding and suggests a direction for rational protein design in the future.

Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation.

BACKGROUND: Lignocellulose is the most abundant natural biomass resource for the production of biofuels and other chemicals. The efficient degradation of cellulose by cellulases is a critical step for the lignocellulose bioconversion. Understanding the structure-catalysis relationship is vital for rational design of more stable and highly active enzymes. Glycoside hydrolase (GH) family 5 is the largest and most functionally diverse group of cellulases, with a conserved TIM barrel structure. The important roles of the various loop regions of GH5 enzymes in catalysis, however, remain poorly understood. RESULTS: In the present study, we investigated the relationship between the loops surrounding active site architecture and its catalytic efficiency, taking TfCel5A, an enzyme from GH5_2 subfamily of Thermobifida fusca, as an example. Large-scale computational simulations and site-directed mutagenesis experiments revealed that three loops (loop 8, 3, and 7) around active cleft played diverse roles in substrate binding, intermediate formation, and product release, respectively. The highly flexible and charged residue triad of loop 8 was responsible for capturing the ligand into the active cleft. Severe fluctuation of loop 3 led to the distortion of sugar conformation at the - 1 subsite. The wobble of loop 7 might facilitate product release, and the enzyme activity of the mutant Y361W in loop 7 was increased by approximately 40%. CONCLUSION: This study unraveled the vital roles of loops in active site architecture and provided new insights into the catalytic mechanism of the GH5_2 cellulases.

Molecular Insights to the Structure-Interaction Relationships of Human Proton-Coupled Oligopeptide Transporters (PepTs).

Human proton-coupled oligopeptide transporters (PepTs) are important membrane influx transporters that facilitate the cellular uptake of many drugs including ACE inhibitors and antibiotics. PepTs mediate the absorption of di- and tri-peptides from dietary proteins or gastrointestinal secretions, facilitate the reabsorption of peptide-bound amino acids in the kidney, and regulate neuropeptide homeostasis in extracellular fluids. PepT1 and PepT2 have been the most intensively investigated of all PepT isoforms. Modulating the interactions of PepTs and their drug substrates could influence treatment outcomes and adverse effects with certain therapies. In recent studies, topology models and protein structures of PepTs have been developed. The aim of this review was to summarise the current knowledge regarding structure-interaction relationships (SIRs) of PepTs and their substrates as well as the potential applications of this information in therapeutic optimisation and drug development. Such information may provide insights into the efficacy of PepT drug substrates in patients, mechanisms of drug-drug/food interactions and the potential role of PepTs targeting in drug design and development strategies.

Molecular dynamics simulations identify the topological weak spots of a protease CN2S8A.

Thermophilic enzymes are highly desired in industrial applications due to their efficient catalytic activity at high temperature. However, most enzymes exhibit inferior thermostability and it remains challenging to identify the optimal sites for designing mutations to improve protein stability. To tackle this issue, we integrated topological analysis and all-atom molecular dynamics simulations to efficiently pinpoint the thermally-unstable regions in protein structures. Using a protease CN2S8A as the model, we analyzed the intramolecular hydrogen bonding interactions between adjacent secondary structure elements, and then identified the topological weak spots of CN2S8A where weak hydrogen bonding interactions were formed. To examine the role of these sites in protein structural stability, we designed three virtual mutations at different weak spots and characterized the effects of these mutations on the structural properties of CN2S8A. The results showed that all three mutations increased the protein structural stability. In conclusion, these findings provide a novel method to identify the topological weak spots of proteins, with implications in the rational design of biocatalysts with superior thermostability.

Lipid A Modification and Metabolic Adaptation in Polymyxin-Resistant, New Delhi Metallo-ß-Lactamase-Producing Klebsiella pneumoniae.

Polymyxins are last-line antibiotics employed against multidrug-resistant (MDR) Klebsiella pneumoniae. Worryingly, polymyxin resistance is rapidly on the rise globally. Polymyxins initially target lipid A of lipopolysaccharides (LPSs) in the cell outer membrane (OM), causing disorganization and cell lysis. While most studies focus on how genetic variations confer polymyxin resistance, the mechanisms of membrane remodeling and metabolic changes in polymyxin-resistant strains remain unclear, thus hampering the development of effective therapies to treat severe K. pneumoniae infections. In the present study, lipid A profiling, OM lipidomics, genomics, and metabolomics were integrated to elucidate the global mechanisms of polymyxin resistance and metabolic adaptation in a polymyxin-resistant strain (strain S01R; MIC of >128 mg/L) obtained from K. pneumoniae strain S01, a polymyxin-susceptible (MIC of 2 mg/L), New Delhi metallo-ß-lactamase (NDM)-producing MDR clinical isolate. Genomic analysis revealed a novel in-frame deletion at position V258 of PhoQ in S01R, potentially leading to lipid A modification with 4-amino-4-deoxy-l-arabinose (L-Ara4N) despite the absence of polymyxin B. Comparative metabolomic analysis revealed slightly elevated levels of energy production and amino acid metabolism in S01R compared to their levels in S01. Exposure to polymyxin B (4 mg/L for S01 and 512 mg/L for S01R) substantially altered energy, nucleotide, and amino acid metabolism and resulted in greater accumulation of lipids in both strains. Furthermore, the change induced by polymyxin B treatment was dramatic at both 1 and 4 h in S01 but only significant at 4 h in S01R. Overall, profound metabolic adaptation was observed in S01R following polymyxin B treatment. These findings contribute to our understanding of polymyxin resistance mechanisms in problematic NDM-producing K. pneumoniae strains and may facilitate the discovery of novel therapeutic targets. IMPORTANCE Antimicrobial resistance (AMR) is a major threat to global health. The emergence of resistance to the polymyxins that are the last line of defense in so-called Gram-negative "superbugs" has further increased the urgency to develop novel therapies. There are frequent outbreaks of K. pneumoniae infections in hospitals being reported, and polymyxin usage is increasing remarkably. Importantly, the polymyxin-resistant K. pneumoniae strains are imposing more severe consequences to health systems. Using metabolomics, lipid A profiling, and outer membrane lipidomics, our findings reveal (i) changes in the pentose phosphate pathway and amino acid and nucleotide metabolism in a susceptible strain following polymyxin treatment and (ii) how cellular metabolism, lipid A modification, and outer membrane remodeling were altered in K. pneumoniae following the acquisition of polymyxin resistance. Our study provides, for the first time, mechanistic insights into metabolic responses to polymyxin treatment in a multidrug-resistant, NDM-producing K. pneumoniae clinical isolate with acquired polymyxin resistance. Overall, these results will assist in identifying new therapeutic targets to combat and prevent polymyxin resistance.

Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase.

Diterpene synthase VenA is responsible for assembling venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. VenA also demonstrates substrate promiscuity by accepting geranyl pyrophosphate and farnesyl pyrophosphate as alternative substrates. Herein, we report the crystal structures of VenA in both apo form and holo form in complex with a trinuclear magnesium cluster and pyrophosphate group. Functional and structural investigations on the atypical 115DSFVSD120 motif of VenA, versus the canonical Asp-rich motif of DDXX(X)D/E, reveal that the absent second Asp of canonical motif is functionally replaced by Ser116 and Gln83, together with bioinformatics analysis identifying a hidden subclass of type I microbial terpene synthases. Further structural analysis, multiscale computational simulations, and structure-directed mutagenesis provide significant mechanistic insights into the substrate selectivity and catalytic promiscuity of VenA. Finally, VenA is semi-rationally engineered into a sesterterpene synthase to recognize the larger substrate geranylfarnesyl pyrophosphate.

Data-driven strategies for the computational design of enzyme thermal stability: trends, perspectives, and prospects.

Thermal stability is one of the most important properties of enzymes, which sustains life and determines the potential for the industrial application of biocatalysts. Although traditional methods such as directed evolution and classical rational design contribute greatly to this field, the enormous sequence space of proteins implies costly and arduous experiments. The development of enzyme engineering focuses on automated and efficient strategies because of the breakthrough of high-throughput DNA sequencing and machine learning models. In this review, we propose a data-driven architecture for enzyme thermostability engineering and summarize some widely adopted datasets, as well as machine learning-driven approaches for designing the thermal stability of enzymes. In addition, we present a series of existing challenges while applying machine learning in enzyme thermostability design, such as the data dilemma, model training, and use of the proposed models. Additionally, a few promising directions for enhancing the performance of the models are discussed. We anticipate that the efficient incorporation of machine learning can provide more insights and solutions for the design of enzyme thermostability in the coming years.

Molecular Dynamics Simulations Reveal the Conformational Transition of GH33 Sialidases.

Sialidases are increasingly used in the production of sialyloligosaccharides, a significant component of human milk oligosaccharides. Elucidating the catalytic mechanism of sialidases is critical for the rational design of better biocatalysts, thereby facilitating the industrial production of sialyloligosaccharides. Through comparative all-atom molecular dynamics simulations, we investigated the structural dynamics of sialidases in Glycoside Hydrolase family 33 (GH33). Interestingly, several sialidases displayed significant conformational transition and formed a new cleft in the simulations. The new cleft was adjacent to the innate active site of the enzyme, which serves to accommodate the glycosyl acceptor. Furthermore, the residues involved in the specific interactions with the substrate were evolutionarily conserved in the whole GH33 family, highlighting their key roles in the catalysis of GH33 sialidases. Our results enriched the catalytic mechanism of GH33 sialidases, with potential implications in the rational design of sialidases.

Gasdermin E regulates the stability and activation of EGFR in human non-small cell lung cancer cells.

BACKGROUND: Lung cancer is the most lethal malignancy, with non-small cell lung cancer (NSCLC) being the most common type (~ 85%). Abnormal activation of epidermal growth factor receptor (EGFR) promotes the development of NSCLC. Chemoresistance to tyrosine kinase inhibitors, which is elicited by EGFR mutations, is a key challenge for NSCLC treatment. Therefore, more thorough understanding of EGFR expression and dynamics are needed. METHODS: Human non-small cell lung cancer cells and HEK293FT cells were used to investigate the molecular mechanism of gasdermin E (GSDME) regulating EGFR stability by Western blot analysis, immunoprecipitation and immunofluorescence. GSDME and EGFR siRNAs or overexpression plasmids were used to characterize the functional role of GSDME and EGFR in vitro. EdU incorporation, CCK-8 and colony formation assays were used to determine the proliferation ability of non-small cell lung cancer cells. RESULTS: GSDME depletion reduced the proliferation of non-small cell lung cancer cells in vitro. Importantly, both GSDME-full length (GSDME-FL) and GSDME-N fragment physically interacted with EGFR. GSDME interacted with cytoplasmic fragment of EGFR. GSDME knockdown inhibited EGFR dimerization and phosphorylation at tyrosine 1173 (EGFRY1173), which activated ERK1/2. GSDME knockdown also promoted phosphorylation of EGFR at tyrosine 1045 (EGFRY1045) and its degradation. CONCLUSION: These results indicate that GSDME-FL increases the stability of EGFR, while the GSDME N-terminal fragment induces EGFR degradation. The GSDME-EGFR interaction plays an important role in non-small cell lung cancer development, reveal a previously unrecognized link between GSDME and EGFR stability and offer new insight into cancer pathogenesis. Video abstract.

Critical Role of Position 10 Residue in the Polymyxin Antimicrobial Activity.

Polymyxins (polymyxin B and colistin) are lipopeptide antibiotics used as a last-line treatment for life-threatening multidrug-resistant (MDR) Gram-negative bacterial infections. Unfortunately, their clinical use has been affected by dose-limiting toxicity and increasing resistance. Structure-activity (SAR) and structure-toxicity (STR) relationships are paramount for the development of safer polymyxins, albeit very little is known about the role of the conserved position 10 threonine (Thr) residue in the polymyxin core scaffold. Here, we synthesized 30 novel analogues of polymyxin B1 modified explicitly at position 10 and examined the antimicrobial activity against Gram-negative bacteria and in vivo toxicity and performed molecular dynamics simulations with bacterial outer membranes. For the first time, this study revealed the stereochemical requirements and role of the ß-hydroxy side chain in promoting the correctly folded conformation of the polymyxin that drives outer membrane penetration and antibacterial activity. These findings provide essential information for developing safer and more efficacious new-generation polymyxin antibiotics.

An Intelligent Strategy with All-Atom Molecular Dynamics Simulations for the Design of Lipopeptides against Multidrug-Resistant Pseudomonas aeruginosa.

Multidrug-resistant Gram-negative bacteria seriously threaten modern medicine due to the lack of efficacious therapeutic options. Their outer membrane (OM) is an essential protective fortress to exclude many antibiotics. Unfortunately, current structural biology methods are not able to resolve the membrane structure and it is difficult to examine the specific interaction between the OM and small molecules. These limitations hinder mechanistic understanding of antibiotic penetration through the OM and antibiotic discovery. Here, we developed biologically relevant OM models by quantitatively determining membrane lipidomics of Pseudomonas aeruginosa and elucidated how lipopolysaccharide modifications and OM vesicles mediated resistance to polymyxins. Supported by chemical biology and pharmacological assays, our multiscale molecular dynamics simulations provide an intelligent platform to quantify the membrane-penetrating thermodynamics of peptides and predict their antimicrobial activity. Through experimental validations with our in-house polymyxin analogue library, our computational strategy may have significant potential in accelerating the discovery of lipopeptides against bacterial "superbugs".

Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity.

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.

A synthetic lipopeptide targeting top-priority multidrug-resistant Gram-negative pathogens.

The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

Molecular Insights into the Assembly and Functional Diversification of Typhoid Toxin.

Typhoid toxin is an A2B5 protein toxin and an important virulence factor for the human-adapted bacterial pathogen Salmonella enterica serovar Typhi, the causative agent of typhoid fever. Typhoid toxin contains two enzymatic subunits, PltA and CdtB, which dock onto a pentameric delivery platform composed of the protein PltB. It was recently reported that the same enzymatic subunits can assemble with a different delivery platform composed of the protein PltC, forming a distinct version of typhoid toxin. However, the differences in structure and receptor specificity between the PltC and PltB typhoid toxins remain unknown. Here, we determined atomic-level structures of the pentameric PltC subunit, the fully assembled PltC typhoid toxin, and the PltC pentamers in complex with glycan receptors. Biochemical and structural analyses indicate that PltB and PltC are unable to form heteromeric delivery complexes due to electrostatic repulsion at the subunit interface and thus form separate toxins only. We further observed that, despite low sequence similarity between PltB and PltC, they interact with PltA in a similar manner but that PltC exhibits stronger electrostatic interactions with PltA, enabling it to outcompete PltB in toxin assembly. The ligand-bound atomic structures of PltC show an additional glycan binding site not found in PltB and glycan array analysis indicates that PltB and PltC exhibit significant differences in glycan binding specificity. Collectively, this study offers atomic-level insights into how S. Typhi produces two distinct versions of typhoid toxin, thereby generating functional diversity in this key virulence factor. IMPORTANCE Typhoid fever is a devastating disease that kills more than 115,000 people every year and is caused by Salmonella Typhi. Typhoid toxin, exclusively produced by S. Typhi, was demonstrated to be responsible for the pathogenesis of typhoid fever. Typhoid toxin consists of a pentameric delivery B subunit to transport the catalytic A subunits into the host cell through binding of the glycan receptors. Recent study shows that S. Typhi encodes two homologous delivery B subunits that are able to associate with the same active subunits to produce alternative toxins with distinct functional characteristics. Here, we show that the two delivery subunits can form only homopentameric delivery platforms that compete to associate with typhoid toxin's active subunits and that the two resulting toxins have distinct glycan-binding properties that confer distinct functional traits. These findings highlight the unique assembly and functional diversification of typhoid toxins.

Docking-guided rational engineering of a macrolide glycosyltransferase glycodiversifies epothilone B.

Glycosyltransferases typically display acceptor substrate flexibility but more stringent donor specificity. BsGT-1 is a highly effective glycosyltransferase to glycosylate macrolides, including epothilones, promising antitumor compounds. Here, we show that BsGT-1 has three major regions significantly influencing the glycodiversification of epothilone B based on structural molecular docking, "hot spots" alanine scanning, and site saturation mutagenesis. Mutations in the PSPG-like motif region and the C2 loop region are more likely to expand donor preference; mutations of the flexible N3 loop region located at the mouth of the substrate-binding cavity produce novel epothilone oligosaccharides. These "hot spots" also functioned in homologues of BsGT-1. The glycosides showed significantly enhanced water solubility and decreased cytotoxicity, although the glycosyl appendages of epothilone B also reduced drug permeability and attenuated antitumor efficacy. This study laid a foundation for the rational engineering of other GTs to synthesize valuable small molecules.

Comparative molecular dynamics simulations identify a salt-sensitive loop responsible for the halotolerant activity of GH5 cellulases.

Halotolerant glycoside hydrolases (GH) have broad application potentials in biorefinery industries. Elucidating the structure-activity relationship underlying the halotolerant catalysis is essential to design superior biocatalysts. Here, we performed molecular dynamics simulations to investigate the structural dynamics of two GH5 cellulases, namely the halotolerant Cel5R and non-halotolerant TfCel5A. Through characterizing the physical properties at different salt concentrations, the results revealed that the overall structures of Cel5R and TfCel5A were marginally affected by the increase in salt concentrations. However, a salt-sensitive loop was identified from both Cel5R and TfCel5A based on its significantly increased flexibility at high salt concentrations. Importantly, compared to TfCel5A the salt-sensitive loop of Cel5R engaged more sodium ions and water molecules around the active site of the enzyme. Besides, the unique residue motif of the salt-sensitive loop in Cel5R formed more intramolecular hydrogen bonds, stabilizing the active architecture of Cel5R at high salt concentrations. Collectively, the structural and dynamic differences may contribute to the various catalytic halotolerance of Cel5R and TfCel5A. These findings provide mechanistic insight into the halotolerant catalysis and will guide the ration design of GH5 cellulases with improved catalytic properties.Communicated by Ramaswamy H. Samy.