Potential natural product 3,4-seco-schitriterpenoids from Kadsura japonica L. as anti-neuroinflammatory agents.

In the present study, the undescribed schitriterpenoids, kadsujanonols A-I (1-9), and eleven reported compounds (10-20) were isolated from K. japonica L. vines. Their structures of 3,4-seco-schitriterpenoids were elucidated mainly by spectroscopic analyses including 1H-, 13C-, and 2D-NMR, IR, HRESIMS spectra. The spatial configurations were determined by the single-crystal X-ray diffraction analysis of kadsujapnonol A (1), 15, 17, and 18, CD data and computational analysis. Furthermore, all isolates were evaluated for the anti-neuroinflammatory activity on LPS-stimulated NO production in BV2 microglial cells and compounds 2, 4, 5, 7, 9, 11, 13-16, and 18 exposed better or comparable suppression abilities than PDTC. Among them, kadlongilactone B (14) showed the best significant inhibiting ability (IC50 = 0.87 µg/mL) and the effect is through the attenuation of the inflammatory transcription factor p65NF-κB. Preliminary structure-activity relationship revealed that δ-lactone at the side chain and 7-member lactone at C-3/C-4, and 3,4:9,10 ring opening are important.

Genetic and Biochemical Characterization of Halogenation and Drug Transportation Genes Encoded in the Albofungin Biosynthetic Gene Cluster.

Albofungin, a hexacyclic aromatic natural product, exhibits broad-spectrum antimicrobial activity. Its biosynthesis, regulation, and resistance remain elusive. Here, we report the albofungin (abf) biosynthetic gene cluster (BGC) from its producing strain Streptomyces tumemacerans JCM5050. The nascent abf BGC encodes 70 putative genes, including regulators, transporters, type II polyketide synthases (PKSs), oxidoreductase, and tailoring enzymes. To validate the intactness and functionality of the BGC, we developed an Escherichia coli-Streptomyces shuttle bacterial artificial chromosome system, whereby the abf BGC was integrated into the genome of a nonproducing host via heterologous conjugation, wherefrom albofungin can be produced, confirming that the BGC is in effect. We then delimited the boundaries of the BGC by means of in vitro CRISPR-Cas9 DNA editing, concluding a minimal but essential 60-kb abf BGC ranging from orfL to abf58. The orfA gene encoding a reduced flavin adenine dinucleotide (FADH2)-dependent halogenase was examined and is capable of transforming albofungin to halogen-substituted congeners in vivo and in vitro. The orfL gene encoding a transporter was examined in vivo. The presence/absence of orfA or orfL demonstrated that the MIC of albofungin is subject to alteration when an extracellular polysaccharide intercellular adhesin was formed. Despite that halogenation of albofungin somewhat increases binding affinity to transglycosylase (TGase), albofungin with/without a halogen substituent manifests similar in vitro antimicrobial activity. Halogenation, however, limits overall dissemination and effectiveness given a high secretion rate, weak membrane permeability, and high hydrophobicity of the resulting products, whereby the functions of orfA and orfL are correlated with drug detoxification/resistance for the first time. IMPORTANCE Albofungin, a natural product produced from Streptomycetes, exhibits bioactivities against bacteria, fungi, and tumor cells. The biosynthetic logic, regulations, and resistance of albofungin remain yet to be addressed. Herein, the minimal albofungin (abf) biosynthetic gene cluster (BGC) from the producing strain Streptomyces tumemacerans JCM5050 was precisely delimited using the Escherichia coli-Streptomyces shuttle bacterial artificial chromosome system, of which the gene essentiality was established in vivo and in vitro. Next, we characterized two genes orfA and orfL encoded in the abf BGC, which act as a reduced flavin adenine dinucleotide (FADH2)-dependent halogenase and an albofungin-congeners transporter, respectively. While each testing microorganism exhibited different sensitivities to albofungins, the MIC values of albofungins against testing strains with/without orfA and/or orfL were subject to considerable changes. Halogen-substituted albofungins mediated by OrfA manifested overall compromised dissemination and effectiveness, revealing for the first time that two functionally distinct proteins OrfA and OrfL are associated together, exerting a novel "belt and braces" mechanism in antimicrobial detoxification/resistance.

Labeling and Characterization of Phenol-Containing Glycopeptides Using Chemoselective Probes with Isotope Tags.

Secondary metabolites are structurally diverse natural products (NPs) and have been widely used for medical applications. Developing new tools to enrich NPs can be a promising solution to isolate novel NPs from the native and complex samples. Here, we developed native and deuterated chemoselective labeling probes to target phenol-containing glycopeptides by the ene-type labeling used in proteomic research. The clickable azido-linker was included for further biotin functionalization to facilitate the enrichment of labeled substrates. Afterward, our chemoselective method, in conjunction with LC-MS and MSn analysis, was demonstrated in bacterial cultures. A vancomycin-related phenol-containing glycopeptide was labeled and characterized by our labeling strategy, showing its potential in glycopeptide discovery in complex environments.

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto-Transferring Reactions.

Transketolase (TK) catalyzes a reversible transfer of a two-carbon (C2 ) unit between phosphoketose donors and phosphoaldose acceptors, for which the group-transfer reaction that follows a one- or two-electron mechanism and the force that breaks the C2"-C3" bond of the ketose donors remain unresolved. Herein, we report ultrahigh-resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group-transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme-catalyzed non-Kekulé diradical cofactor brings about the C2"-C3" bond cleavage/formation for the C2 -unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated.

The Mesomeric Effect of Thiazolium on non-Kekulé Diradicals in Pichia stipitis Transketolase.

It is theoretically plausible that thiazolium mesomerizes to congeners other than carbene in a low effective dielectric binding site; especially given the energetics and uneven electronegativity of carbene groups. However, such a phenomenon has never been reported. Nine crystal structures of transketolase obtained from Pichia stipitis (TKps) are reported with subatomic resolution, where thiazolium displays an extraordinary ring-bending effect. The bent thiazolium congeners correlate with non-Kekulé diradicals because there is no gain or loss of electrons. In conjunction with biophysical and biochemical analyses, it is concluded that ring bending is a result of tautomerization of thiazolium with its non- Kekulé diradicals, exclusively in the binding site of TKps. The chemophysical properties of these thiazolium mesomers may account for the great variety of reactivities carried out by thiamine-diphosphate-containing (ThDP) enzymes. The stability of ThDP in living systems can be regulated by the levels of substrates, and hydration and dehydration, as well as diradical-mediated oxidative degradation.

Nanodiamonds as Nucleating Agents for Protein Crystallization.

Nanodiamond (ND) is a carbon-based nanomaterial with potential for a wide range of biological applications. One of such applications is to facilitate the nucleation of protein crystals in aqueous solution. Here, we show that NDs (nominal diameters of 30 and 100 nm) after surface oxidation in air and subsequent treatment in strong acids are useful as heterogeneous nucleating agents for protein crystallization. Tested with lysozyme, ribonuclease A, proteinase K, and catalase, the nanomaterials in either aggregate or film form are found to be able to increase the crystallization efficiency of all proteins. Particularly, for 30 nm NDs, the films with an area of ∼2 mm2 can effectively induce the crystallization of lysozyme at a concentration as low as 5 mg/mL. The efficiency can be further improved by adding preformed protein clusters (∼300 nm in diameter) as inherent nucleation precursors, as demonstrated for ribonuclease A. This combined approach is easy to implement, highly compatible with existing technologies, and can be applied to other protein samples as well.

Prophylactic administration of fucoidan represses cancer metastasis by inhibiting vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in Lewis tumor-bearing mice.

Fucoidan, a heparin-like sulfated polysaccharide, is rich in brown algae. It has a wide assortment of protective activities against cancer, for example, induction of hepatocellular carcinoma senescence, induction of human breast and colon carcinoma apoptosis, and impediment of lung cancer cells migration and invasion. However, the anti-metastatic mechanism that fucoidan exploits remains elusive. In this report, we explored the effects of fucoidan on cachectic symptoms, tumor development, lung carcinoma cell spreading and proliferation, as well as expression of metastasis-associated proteins in the Lewis lung carcinoma (LLC) cells-inoculated mice model. We discovered that administration of fucoidan has prophylactic effects on mitigation of cachectic body weight loss and improvement of lung masses in tumor-inoculated mice. These desired effects are attributed to inhibition of LLC spreading and proliferation in lung tissues. Fucoidan also down-regulates expression of matrix metalloproteinases (MMPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and vascular endothelial growth factor (VEGF). Moreover, the tumor-bearing mice supplemented with fucoidan indeed benefit from an ensemble of the chemo-phylacticity. The fact is that fucoidan significantly decreases viability, migration, invasion, and MMPs activities of LLC cells. In summary, fucoidan is suitable to act as a chemo-preventative agent for minimizing cachectic symptoms as well as inhibiting lung carcinoma metastasis through down-regulating metastatic factors VEGF and MMPs.

Insights into the binding specificity and catalytic mechanism of N-acetylhexosamine 1-phosphate kinases through multiple reaction complexes.

Utilization of N-acetylhexosamine in bifidobacteria requires the specific lacto-N-biose/galacto-N-biose pathway, a pathway differing from the Leloir pathway while establishing symbiosis between humans and bifidobacteria. The gene lnpB in the pathway encodes a novel hexosamine kinase NahK, which catalyzes the formation of N-acetylhexosamine 1-phosphate (GlcNAc-1P/GalNAc-1P). In this report, seven three-dimensional structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP from both Bifidobacterium longum (JCM1217) and B. infantis (ATCC15697) were solved at resolutions of 1.5-2.2 Å. NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The NahK structures presented here represent the first multiple reaction complexes of the enzyme. This structural information reveals the molecular basis for the recognition of the given substrates and products, GlcNAc/GalNAc, GlcNAc-1P/GalNAc-1P, ATP/ADP and Mg(2+), and provides insights into the catalytic mechanism, enabling NahK and mutants thereof to form a choice of biocatalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.

Structure and mechanism of a nonhaem-iron SAM-dependent C-methyltransferase and its engineering to a hydratase and an O-methyltransferase.

In biological systems, methylation is most commonly performed by methyltransferases (MTs) using the electrophilic methyl source S-adenosyl-L-methionine (SAM) via the S(N)2 mechanism. (2S,3S)-ß-Methylphenylalanine, a nonproteinogenic amino acid, is a building unit of the glycopeptide antibiotic mannopeptimycin. The gene product of mppJ from the mannopeptimycin-biosynthetic gene cluster is the MT that methylates the benzylic C atom of phenylpyruvate (Ppy) to give ßMePpy. Although the benzylic C atom of Ppy is acidic, how its nucleophilicity is further enhanced to become an acceptor for C-methylation has not conclusively been determined. Here, a structural approach is used to address the mechanism of MppJ and to engineer it for new functions. The purified MppJ displays a turquoise colour, implying the presence of a metal ion. The crystal structures reveal MppJ to be the first ferric ion SAM-dependent MT. An additional four structures of binary and ternary complexes illustrate the molecular mechanism for the metal ion-dependent methyltransfer reaction. Overall, MppJ has a nonhaem iron centre that bind, orients and activates the α-ketoacid substrate and has developed a sandwiched bi-water device to avoid the formation of the unwanted reactive oxo-iron(IV) species during the C-methylation reaction. This discovery further prompted the conversion of the MT into a structurally/functionally unrelated new enzyme. Through stepwise mutagenesis and manipulation of coordination chemistry, MppJ was engineered to perform both Lewis acid-assisted hydration and/or O-methyltransfer reactions to give stereospecific new compounds. This process was validated by six crystal structures. The results reported in this study will facilitate the development and design of new biocatalysts for difficult-to-synthesize biochemicals.

Multiple complexes of long aliphatic N-acyltransferases lead to synthesis of 2,6-diacylated/2-acyl-substituted glycopeptide antibiotics, effectively killing vancomycin-resistant enterococcus.

Teicoplanin A2-2 (Tei)/A40926 is the last-line antibiotic to treat multidrug-resistant Gram-positive bacterial infections, e.g., methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). This class of antibiotics is powered by the N-acyltransferase (NAT) Orf11*/Dbv8 through N-acylation on glucosamine at the central residue of Tei/A40926 pseudoaglycone. The NAT enzyme possesses enormous value in untapped applications; its advanced development is hampered largely due to a lack of structural information. In this report, we present eight high-resolution X-ray crystallographic unary, binary, and ternary complexes in order to decipher the molecular basis for NAT's functionality. The enzyme undergoes a multistage conformational change upon binding of acyl-CoA, thus allowing the uploading of Tei pseudoaglycone to enable the acyl-transfer reaction to take place in the occlusion between the N- and C-halves of the protein. The acyl moiety of acyl-CoA can be bulky or lengthy, allowing a large extent of diversity in new derivatives that can be formed upon its transfer. Vancomycin/synthetic acyl-N-acetyl cysteamine was not expected to be able to serve as a surrogate for an acyl acceptor/donor, respectively. Most strikingly, NAT can catalyze formation of 2-N,6-O-diacylated or C6→C2 acyl-substituted Tei analogues through an unusual 1,4-migration mechanism under stoichiometric/solvational reaction control, wherein selected representatives showed excellent biological activities, effectively counteracting major types (VanABC) of VRE.

Biosynthesis of streptolidine involved two unexpected intermediates produced by a dihydroxylase and a cyclase through unusual mechanisms.

Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a ß-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe(II) -dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2 -L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH)2 -L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment.

Rejuvenation of Meropenem by Conjugation with Tilapia Piscidin-4 Peptide Targeting NDM-1 Escherichia coli.

Gram-negative pathogens that produce ß-lactamases pose a serious public health threat as they can render ß-lactam antibiotics inactive via hydrolysis. This action contributes to the waning effectiveness of clinical antibiotics and creates an urgent need for new antimicrobials. Antimicrobial peptides (AMPs) exhibiting multimodal functions serve as a potential source in spite of a few limitations. Thus, the conjugation of conventional antibiotics with AMPs may be an effective strategy to leverage the advantages of each component. In this study, we conjugated meropenem to the AMP Tilapia piscidin 4 (TP4) using a typical coupling reaction. The conjugate was characterized by using HPLC-MS, HR-MS, and MS-MS fragmentation analysis. It was then evaluated in terms of antibacterial potency, hemolysis, and cytotoxicity toward RAW264.7 and CCD-966SK cell lines. The conjugation of meropenem with TP4 significantly reduced the cytotoxicity compared to TP4. Conjugation of unprotected TP4 with meropenem resulted in cross-linking at the N-terminal and lysine sites. The structural activity relationship of the two isomers of the TP4-meropenem conjugate was investigated. Both the isomers showed notable antibacterial activities against NDM-1 Escherichia coli and reduced red blood cell hemolysis as compared to TP4. Lysine conjugate (TP4-K-Mero) showed lesser hemolysis than the N-terminal conjugate (TP4-N-Mero). Molecular modeling further revealed that the conjugates can bind to lipopolysaccharides and inhibit NDM-1 ß-lactamase. Together, these data show that conjugation of antibiotics with AMP can be a feasible approach to increase the therapeutic profile and effectively target multidrug-resistant pathogens. Furthermore, antibiotic conjugation at different AMP sites tends to show unique biological properties.

Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds.

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.

Kaguacidine A: a novel spirohydantoin-containing cucurbitane glycoside from vines of Momordica charantia L.

The spirohydantoin-containing cucurbitane-type triterpenoid, kaguacidine A (1), was isolated and purified from 95% ethanol extract of vines of Momordica charantia L. (Cucurbitaceae). Its unprecedented chemical structure, a spirohydantoin substituent at C-23 of cucurbitane, was elucidated by extensive spectroscopic analyses, including HRESIMS, IR, optical rotation, 1 D- and 2 D-NMR spectra. The possible biosynthetic pathway is deduced and may be attributed to the metabolic activity of microbial symbionts in M. charantia L. Compound 1 was evaluated for anti-inflammatory activity against LPS-induced NO production in RAW 264.7 cells and anti-proliferative activity against four cancer cell lines, including HEp-2, MCF-7, Hep-G2, and WiDr. Compound 1 showed moderate anti-inflammatory activity with an IC50 value of 18.5 ± 0.4 µg/mL and weak anti-proliferative activity against MCF-7, HEp-2, Hep-G2, and WiDr with IC50 values of >40, 33.8 ± 0.6, 31.0 ± 0.7, and 27.0 ± 0.7 µM, respectively.

N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry.

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

Anti-inflammatory effect of euphane- and tirucallane-type triterpenes isolated from the traditional herb Euphorbia neriifolia L.

The Euphorbiaceae plant Euphorbia neriifolia L. is distributed widely in India, Thailand, Southeastern China, and Taiwan and used as a carminative and expectorant to treat several inflammation-related diseases, such as gonorrhoea, asthma, and cancer. In the course of our search for potential anti-inflammatory agents from the titled plant, 11 triterpenes from the stem of E. neriifolia were isolated and reported in our previous endeavor. Given its rich abundance in triterpenoids, the ethanolic extract in this follow-up exploration has led to the isolation of additional eight triterpenes, including six new euphanes-neritriterpenols H and J-N (1 and 3-7)-one new tirucallane, neritriterpenol I (2), and a known compound, 11-oxo-kansenonol (8). Their chemical structures were elucidated on the basis of spectroscopic data, including 1D- and 2D NMR, and HRESIMS spectra. The absolute stereochemistry of neritriterpenols was determined by single-crystal X-ray diffraction analysis, ICD spectra, and DP4+ NMR data calculations. Compounds 1-8 were also evaluated for their anti-inflammatory activity by using lipopolysaccharide (LPS)-stimulated IL-6 and TNF-α on RAW 264.7 macrophage cells. Intriguingly, the euphane-type triterpenes (1 and 3-8) showed an inhibitory effect on LPS-induced IL-6 but not on TNF-α, while tirucallane-type triterpene 2 showed strong inhibition on both IL-6 and TNF-α.

Chain elongation and cyclization in type III PKS DpgA.

Chain elongation and cyclization of precursors of dihydroxyphenylacetyl-CoA (DPA-CoA) catalyzed by the bacterial type III polyketide synthase DpgA were studied. Two labile intermediates, di- and tri-ketidyl-CoA (DK- and TK-CoA), were proposed and chemically synthesized. In the presence of DpgABD, each of these with [(13)C(3)]malonyl-CoA (MA-CoA) was able to form partially (13)C-enriched DPA-CoA. By NMR and MS analysis, the distribution of (13)C atoms in the partially (13)C-enriched DPA-CoA shed light on how the polyketide chain elongates and cyclizes in the DpgA-catalyzed reaction. Polyketone intermediates elongate in a manner different from that which had been believed: two molecules of DK-CoA, or one DK-CoA plus one acetoacetyl-CoA (AA-CoA), but not two molecules of AA-CoA can form one molecule of DPA-CoA. As a result, polyketidyl-CoA serves as both the starter and extender, whereas polyketone-CoA without the terminal carboxyl group can only act as an extender. The terminal carboxyl group is crucial for the cyclization that likely takes place on CoA.

Fucoidan from Sargassum hemiphyllum inhibits infection and inflammation of Helicobacter pylori.

Having infected by Helicobacter pylori, the infection often leads to gastritis, gastric ulcer, or even gastric cancer. The disease is typically treated with antibiotics as they used to effectively inhibit or kill H. pylori, thus reducing the incidence of gastric adenoma and cancer to significant extent. H. pylori, however, has developed drug resistance to many clinically used antibiotics over the years, highlighting the crisis of antibiotic failure during the H. pylori treatment. We report here that the fucoidan from Sargassum hemiphyllum can significantly reduce the infection of H. pylori without developing to drug resistance. Fucoidan appears to be a strong anti-inflammation agent as manifested by the RAW264.7 cell model examination. Fucoidan can prohibit H. pylori adhesion to host cells, thereby reducing the infection rate by 60%, especially in post treatment in the AGS cell model assay. Mechanistically, fucoidan intervenes the adhesion of BabA and AlpA of H. pylori significantly lowering the total count of H. pylori and the level of IL-6 and TNF-α in vivo. These results all converge on the same fact that fucoidan is an effective agent in a position to protect the stomach from the H. pylori infection by reducing both the total count and induced inflammation.

Intermolecular Interactions of Nucleoside Antibiotic Tunicamycin with On-Target MraYCB-TUN and Off-Target DPAGT1-TUN in the Active Sites Delineated by Quantum Mechanics/Molecular Mechanics Calculations.

Tunicamycin (TUN) is a nucleoside antibiotic with a complex structure comprising uracil, tunicamine sugar, N-acetylglucosamine (GlcNAc), and fatty acyl tail moieties. TUN, known as a canonical inhibitor, blocks vital functions of certain transmembrane protein families, for example, the insect enzyme dolichyl phosphate α-N-acetylglucosaminylphosphotransferase (DPAGT1) of Spodoptera frugiperda and the bacterial enzyme phospho-N-acetylmuramoylpentapeptide translocase (MraYCB) of Clostridium bolteae. Accurate description of protein-drug interactions has an immense impact on structure-based drug design, while the main challenge is to create proper topology and parameter entries for TUN in modeling protein-TUN interactions given the structural complexity. Starting from DPAGT1-TUN and MraYCB-TUN crystal structures, we first sketched these structural complexes on the basis of the CHARMM36 force field and optimized each of them using quantum mechanics/molecular mechanics (QM/MM) calculations. By continuing calculations on the active site (QM region) of each optimized structure, we specified the characteristics of intermolecular interactions contributing to the binding of TUN to each active site by quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses at the M06-2X/6-31G** level. The results outlined that TUN insertion into each active site requires multiple weak, moderate, and strong hydrogen bonds accompanying charge-dipole, dipole-dipole, and hydrophobic interactions among different TUN moieties and adjacent residues. The water-mediated interactions also play central roles in situating the uracil and tunicamine moieties of TUN within the DPAGT1 active site as well as in preserving the uracil-binding pocket in the MraYCB active site. The TUN binds more strongly to DPAGT1 than to MraYCB. The information garnered here is valuable particularly for better understanding mode of action at the molecular level, as it is conducive to developing next generations of nucleoside antibiotics.