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Volatile organic compounds (VOCs) have harmful effects on human health and the environment but detecting low levels of VOCs is challenging due to a lack of reliable biomarkers. However, incorporating gold nanoparticles (Au NPs) into metal-organic frameworks (MOFs) shows promise for VOC detection. In this study, we developed nanoscale Au@UiO-66 that exhibited surface-enhanced Raman scattering (SERS) activity even at very low levels of toluene vapors (down to 1.0 ppm) due to the thickness of the shell and strong π-π interactions between benzenyl-type linkers and toluene. The UiO-66 shell also increased the thermal stability of the Au NPs, preventing aggregation up to 550 °C. This development may be useful for sensitive detection of VOCs for environmental protection purposes.
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Chikungunya virus (CHIKV) has repeatedly spread via the bite of an infected mosquito and affected more than 100 countries. The disease poses threats to public health and the economy in the infected locations. Many efforts have been devoted to identifying compounds that could inhibit CHIKV. Unfortunately, successful clinical candidates have not been found yet. Computations through the simulating recognition process were performed on complexation of the nsP3 protein of CHIKV with the structures of triply conjugated drug lead candidates. The outcomes provided the aid on rational design of functionalized quinazoline-(α-substituted coumarin)-arylsulfonate compounds to inhibit CHIKV in Vero cells. The molecular docking studies showed a void space around the ß carbon atom of coumarin when a substituent was attached at the α position. The formed vacancy offered a good chance for a Michael addition to take place owing to steric and electronic effects. The best conjugate containing a quinazolinone moiety exhibited potency with EC50 = 6.46 µM, low toxicity with CC50 = 59.7 µM, and the selective index (SI) = 9.24. Furthermore, the corresponding 4-anilinoquinazoline derivative improved the anti-CHIKV potency to EC50 = 3.84 µM, CC50 = 72.3 µM, and SI = 18.8. The conjugate with 4-anilinoquinazoline exhibited stronger binding affinity towards the macro domain than that with quinazolinone via hydrophobic and hydrogen bond interactions.
Asunto(s)
Virus Chikungunya , Animales , Antivirales/química , Arilsulfonatos/metabolismo , Arilsulfonatos/farmacología , Chlorocebus aethiops , Diseño Asistido por Computadora , Cumarinas/farmacología , Simulación del Acoplamiento Molecular , Quinazolinas/metabolismo , Quinazolinas/farmacología , Quinazolinonas/farmacología , Células Vero , Replicación ViralRESUMEN
Applying metal-organic frameworks (MOFs) on the surface of other materials to form multifunctional materials has recently attracted great attention; however, directing the MOF overgrowth is challenging due to the orders of magnitude differences in structural dimensions. In this work, we developed a universal strategy to mediate MOF growth on the surface of metal nanoparticles (NPs), by taking advantage of the dynamic nature of weakly adsorbed capping agents. During this colloidal process, the capping agents gradually dissociate from the metal surface, replaced in situ by the MOF. The MOF grows to generate a well-defined NP-MOF interface without a trapped capping agent, resulting in a uniform core-shell structure of one NP encapsulated in one single-crystalline MOF nanocrystal with specific facet alignment. The concept was demonstrated by coating ZIF-8 and UiO-66-type MOFs on shaped metal NPs capped by cetyltrimethylammonium surfactants, and the formation of the well-defined NP-MOF interface was monitored by spectroscopies. The defined interface outperforms ill-defined ones generated via conventional methods, displaying a high selectivity to unsaturated alcohols for the hydrogenation of an α,ß-unsaturated aldehyde. This strategy opens a new route to create aligned interfaces between materials with vastly different structural dimensions.
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It has been reported that the biological functions of enzymes could be altered when they are encapsulated in metal-organic frameworks (MOFs) due to the interactions between them. Herein, we probed the interactions of catalase in solid and hollow ZIF-8 microcrystals. The solid sample with confined catalase is prepared through a reported method, and the hollow sample is generated by hollowing the MOF crystals, sealing freestanding enzymes in the central cavities of hollow ZIF-8. During the hollowing process, the samples were monitored by small-angle X-ray scattering (SAXS) spectroscopy, electron microscopy, powder X-ray diffraction (PXRD), and nitrogen sorption. The interfacial interactions of the two samples were studied by infrared (IR) and fluorescence spectroscopy. IR study shows that freestanding catalase has less chemical interaction with ZIF-8 than confined catalase, and a fluorescence study indicates that the freestanding catalase has lower structural confinement. We have then carried out the hydrogen peroxide degradation activities of catalase at different stages and revealed that the freestanding catalase in hollow ZIF-8 has higher activity.
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Estructuras Metalorgánicas , Catalasa , Enzimas Inmovilizadas , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
We present a dielectric spectroscopy study of dipolar dynamics in the hydrated UiO-66(Zr) type metal-organic frameworks (MOFs) functionalized with -NH2 and -F groups. Experiments are performed in a broad temperature and frequency ranges allowing us to probe several dipolar relaxations. For both samples at temperature below 220 K, we observe confined supercooled water dynamics, which can be described by the Arrhenius law. At slightly higher temperature, a second less pronounced dipolar relaxation is identified, and its origin is discussed. At even higher temperature, the dielectric permittivity exhibits anomalous increase with increasing temperature due to the proton conductivity. Upon further heating, the permittivity shows a sudden decrease indicating a reversible removal of water molecules. Measurements of the dehydrated samples reveal absence of all three dipolar processes.
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Espectroscopía Dieléctrica , Estructuras Metalorgánicas/química , Compuestos Organometálicos/química , Ácidos Ftálicos/química , Agua/química , Algoritmos , Modelos Químicos , Estructura Molecular , TemperaturaRESUMEN
The syntheses of metal-organic frameworks (MOFs) can be improved through modulated synthesis, synthesis employing precursors, and postsynthetic exchange (PSE) modifications, all of which share ligand exchange as a common and crucial reaction. To date, however, the mechanism of ligand exchange and the underlying principles governing it have remained elusive. Herein, we report energy landscapes for the ligand exchange processes of 1,4-benzenedicarboxylic acid and 2,3,5,6-tetrafluoro-1,4-benzenedicarboxylic acid with Zr6O4(OH)4(OMc)12 (OMc = methacrylate), as calculated using density functional theory (DFT). The rate-limiting step of ligand exchange follows an associative-substitution mechanism catalyzed by protons, consistent with previous kinetic data. Our calculations suggest that the acid catalysis is dependent on the relative basicities of the incoming and outgoing ligands coordinated in the complex, allowing molecular-level rationalization of many seminal MOF syntheses that had previously been interpreted macroscopically. Our results provide new insights for MOF synthesis and new clues for the rational de novo synthesis of MOFs.
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We show that an enzyme maintains its biological function under a wider range of conditions after being embedded in metal-organic framework (MOF) microcrystals via a de novo approach. This enhanced stability arises from confinement of the enzyme molecules in the mesoporous cavities in the MOFs, which reduces the structural mobility of enzyme molecules. We embedded catalase (CAT) into zeolitic imidazolate frameworks (ZIF-90 and ZIF-8), and then exposed both embedded CAT and free CAT to a denature reagent (i.e., urea) and high temperatures (i.e., 80 °C). The embedded CAT maintains its biological function in the decomposition of hydrogen peroxide even when exposed to 6 M urea and 80 °C, with apparent rate constants kobs (s-1) of 1.30 × 10-3 and 1.05 × 10-3, respectively, while free CAT shows undetectable activity. A fluorescence spectroscopy study shows that the structural conformation of the embedded CAT changes less under these denaturing conditions than free CAT.
Asunto(s)
Catalasa/química , Imidazoles/farmacología , Estructuras Metalorgánicas/farmacología , Desplegamiento Proteico/efectos de los fármacos , Zeolitas/farmacología , Catalasa/metabolismo , Imidazoles/química , Estructuras Metalorgánicas/química , Tamaño de la Partícula , Porosidad/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Propiedades de Superficie/efectos de los fármacos , Temperatura , Zeolitas/químicaRESUMEN
Using a simple method, the aldehyde groups of zeolitic imidazolate framework-90 (ZIF-90) nanocrystals were converted into carboxyl, amino, and thiol groups, without affecting the integrity of the framework. Notably, for the first time, correlations between functionality and cytotoxicity are also demonstrated via in vitro cytotoxicity assays. The positive charged aminated-ZIF-90 presumably results in either perturbation of cell membrane, more efficient cell uptake, or both. Therefore, the half-maximal effective (EC50 ) concentration of aminated-ZIF-90 has a higher cytotoxicity of about 30â µg mL(-1) .
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Imidazoles/química , Nanopartículas/química , Zeolitas/química , Zeolitas/toxicidad , Aldehídos/química , Aminación , Imidazoles/toxicidad , Modelos Moleculares , Compuestos de Sulfhidrilo/químicaRESUMEN
A series of new conjugated compounds with a -SCH2- linkage were synthesized by chemical methods from imidazole and coumarin derivatives. The experimental results indicate that of the twenty newly synthesized imidazole-coumarin conjugates, three of them exhibited appealing EC50 values (5.1-8.4 µM) and selective indices >20 against hepatitis C virus. Their potency and selectivity were increased substantially by modification of their structure with two factors: imidazole nucleus with a hydrogen atom at the N(1) position and coumarin nucleus with a substituent, such as Cl, F, Br, Me, and OMe. These guidelines provide valuable information for further development of conjugated compounds as anti-viral agents.
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Cumarinas/síntesis química , Hepacivirus/efectos de los fármacos , Imidazoles/síntesis química , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Cumarinas/química , Cumarinas/farmacología , Hepacivirus/fisiología , Imidazoles/química , Imidazoles/farmacología , Estructura Molecular , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
We develop a new concept to impart new functions to biocatalysts by combining enzymes and metal-organic frameworks (MOFs). The proof-of-concept design is demonstrated by embedding catalase molecules into uniformly sized ZIF-90 crystals via a de novo approach. We have carried out electron microscopy, X-ray diffraction, nitrogen sorption, electrophoresis, thermogravimetric analysis, and confocal microscopy to confirm that the ~10 nm catalase molecules are embedded in 2 µm single-crystalline ZIF-90 crystals with ~5 wt % loading. Because catalase is immobilized and sheltered by the ZIF-90 crystals, the composites show activity in hydrogen peroxide degradation even in the presence of protease proteinase K.
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Biocatálisis , Catalasa/química , Catalasa/metabolismo , Nanoporos , Compuestos Organometálicos/química , Tamaño de la Partícula , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Peróxido de Hidrógeno/metabolismo , Imidazoles/química , Cinética , Modelos Moleculares , Conformación Proteica , Zeolitas/químicaRESUMEN
This study unveils the "green" metal-organic framework (MOF) structuring mechanism by decoding proton transfer in water during ZIF-8 synthesis. Combining in situ small- to wide-angle X-ray scattering, multiscale simulations, and quantum calculations, we reveal that the ZIF-8 early-stage nucleation and crystallization process in aqueous solution unfolds in three distinct stages. In stage I, imidazole ligands replace water in zinc-water cages, triggering an "acidity flip" that promotes proton transfer. This leads to the assembly of structures from single zinc ions to 3D amorphous cluster nuclei. In stage II, amorphous nuclei undergo a critical transformation, evolving into crystalline nuclei and subsequently forming mesoscale-ordered structures and crystallites. The process proceeds until the amorphous precursors are completely consumed, with the transformation kinetics governed by an energy barrier that determines the rate-limiting step. In stage III, stable crystallite nanoparticles form in solution, characterized by a temperature-dependent thermal equilibrium of molecular interactions at the crystal-solution interface. Beyond these core advancements, we explore the influence of encapsulated pepsin and nonencapsulated lysozyme on ZIF-8 formation, finding that their amino acid proton transfer capacity and concentration influence the resulting biomolecule-MOF composite's shape and encapsulation efficiency. The findings contribute to understanding the molecular mechanisms behind biomimetic mineralization and have potential implications for engineering proteins within amorphous MOF nuclei as protein embryo growth sites.
RESUMEN
The ZIF code: ZIF-90 materials were successfully synthesized in an optimized water-based system. The particle size, ranging from micro- to nanoscales, could be controlled by different amounts of polyvinylpyrrolidone (PVP), Zn/imidazole-2-carboxaldehyde ratio and alcohol.
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Imidazoles/química , Agua/química , Zeolitas/química , Alcoholes/química , Tecnología Química Verde , Nanopartículas/química , Tamaño de la Partícula , Povidona/químicaRESUMEN
Highly ordered benzene-bridged periodic mesoporous organosilicas (PMOs) that were functionalized with exceptionally high loadings of carboxylic acid groups (COOH), up to 80â mol % based on silica, have been synthesized and their use as adsorbents for the adsorption of methylene blue (MB), a basic dye pollutant, and for the loading and release of doxorubicin (DOX), an anticancer drug, is demonstrated. These COOH-functionalized benzene-silicas were synthesized by the co-condensation of 1,4-bis(triethoxysilyl) benzene (BTEB) and carboxyethylsilanetriol sodium salt (CES), an organosilane that contained a carboxylic acid group, in the presence of non-ionic oligomeric surfactant Brijâ 76 in acidic medium. The materials thus obtained were characterized by a variety of techniques, including powder X-ray diffraction (XRD), nitrogen-adsorption/desorption isotherms, TEM, and (13)C and (29)Si solid-state NMR spectroscopy. Owing to the exceptionally high loadings of COOH groups, their high surface areas, and possible π-π-stacking interactions, these adsorbents have very high adsorption capacities and extremely rapid adsorption rates for MB removal and for the controlled loading/release of DOX, thus manifesting their great potential for environmental and biomedical applications.
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Derivados del Benceno/química , Ácidos Carboxílicos/química , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Azul de Metileno/análisis , Compuestos de Organosilicio/síntesis química , Adsorción , Espectroscopía de Resonancia Magnética , Azul de Metileno/química , Modelos Químicos , Compuestos de Organosilicio/química , Porosidad , Difracción de Rayos XRESUMEN
MOFs have been effectively used to magnify the triboelectric charge of polymers. However, so far the individual triboelectric properties and charge transfer mechanisms of MOFs haven't been reported. Triboelectric property investigation for selected MOFs show that the main mechanism for MOF triboelectrification in contact with metals is electron transfer.
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A zinc-based metal organic framework, Zn-MOF-74, which has a unique one-dimensional (1D) channel and nanoscale aperture size, was rapidly obtained in 10 min using a de novo mild water-based system at room temperature, which is an example of green and sustainable chemistry. First, catalase (CAT) enzyme was encapsulated into Zn-MOF-74 (denoted as CAT@Zn-MOF-74), and comparative assays of biocatalysis, size-selective protection, and framework-confined effects were investigated. Electron microscopy and powder X-ray diffraction were used for characterization, while electrophoresis and confocal microscopy confirmed the immobilization of CAT molecules inside the single hexagonal MOF crystals at loading of â¼15 wt %. Furthermore, the CAT@Zn-MOF-74 hybrid was exposed to a denaturing reagent (urea) and proteolytic conditions (proteinase K) to evaluate its efficacy. The encapsulated CAT maintained its catalytic activity in the decomposition of hydrogen peroxide (H2O2), even when exposed to 0.05 M urea and proteinase K, yielding an apparent observed rate constant (kobs) of 6.0 × 10-2 and 6.6 × 10-2 s-1, respectively. In contrast, free CAT exhibited sharply decreased activity under these conditions. Additionally, the bioactivity of CAT@Zn-MOF-74 for H2O2 decomposition was over three times better than that of the biocomposites based on zeolitic imidazolate framework 90 (ZIF-90) owing to the nanometer-scaled apertures, 1D channel, and less confinement effects in Zn-MOF-74 crystallites. To demonstrate the general applicability of this strategy, another enzyme, α-chymotrypsin (CHT), was also encapsulated in Zn-MOF-74 (denoted as CHT@Zn-MOF-74) for action against a substrate larger than H2O2. In particular, CHT@Zn-MOF-74 demonstrated a biological function in the hydrolysis of l-phenylalanine p-nitroanilide (HPNA), the activity of ZIF-90-encapsulated CHT was undetectable due to aperture size limitations. Thus, we not only present a rapid eco-friendly approach for Zn-MOF-74 synthesis but also demonstrate the broader feasibility of enzyme encapsulation in MOFs, which may help to meet the increasing demand for their industrial applications.
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Emodin--a major component of Rheum palmatum L.-exerts antiproliferative effects in cancer cells that are regulated by different signaling pathways. Hepatocellular carcinoma has high-incidence rates and is associated with poor prognosis and high mortality rates. This study was designed to evaluate the effects of emodin on human hepatocarcinoma cell viability and investigate its mechanisms of action in Huh7, Hep3B, and HepG2 cells. To define the molecular changes associated with this process, expression profiles were compared in emodin-treated hepatoma cells by cDNA microarray hybridization, quantitative RT-PCRs, and Western blot analysis. G2/M phase arrest was observed in all 3 cell lines. Cell cycle regulatory gene analysis showed increased protein levels of cyclin A, cyclin B, Chk2, Cdk2, and P27 in hepatoma cells after time courses of emodin treatment, and Western blot analysis showed decreased protein levels of Cdc25c and P21. Microarray expression profile data and quantitative PCR revealed that 15 representative genes were associated with emodin treatment response in hepatoma cell lines. The RNA expression levels of CYP1A1, CYP1B1, GDF15, SERPINE1, SOS1, RASD1, and MRAS were upregulated and those of NR1H4, PALMD, and TXNIP were downregulated in all three hepatoma cells. Moreover, at 6h after emodin treatment, the levels of GDF15, CYP1A1, CYP1B1, and CYR61 were upregulated. Here, we show that emodin treatment caused G2/M arrest in liver cancer cells and increased the expression levels of various genes both in mRNA and protein level. It is likely that these genes act as biomarkers for hepatocellular carcinoma therapy.
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Antineoplásicos/farmacología , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/genética , Emodina/farmacología , Neoplasias Hepáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis , Carcinoma Hepatocelular/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Since its discovery in Tanganyika, Africa in 1952, chikungunya virus (CHIKV) outbreaks have occurred in Africa, Asia, Europe, and America. Till now chikungunya fever has spread in nearly 40 countries. Because of lack of effective vaccines and antiviral drugs to intervene this disease, 21 new conjugated compounds were designed and synthesized by coupling of 6,8-dithioguanosine at its C-6 position with 3-(chloromethyl)coumarins bearing an F, Cl, Br, Me, or -OMe substituent through the -SCH2- joint. Meanwhile, an organic "dummy" ligand (e.g., methyl, benzyl, and naphthylmethyl) or a coumarinyl moiety was attached at the C-8 position. By high through-put screening, three of these new conjugates were found to inhibit CHIKV in Vero cells with significant potency (EC50â¯=â¯9.9-13.9⯵M) and showed low toxicity (CC50â¯=â¯96.5-212⯵M). The selectivity index values were 9.37-21.7. Their structure-activity relationship was deduced, which indicates that the coumarin moiety is essential and the presence of a -OMe group enhances the antiviral activity.
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Antivirales/química , Antivirales/farmacología , Virus Chikungunya/efectos de los fármacos , Cumarinas/química , Guanosina/química , Guanosina/farmacología , Animales , Chlorocebus aethiops , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Células VeroRESUMEN
Metal-organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely ß-glucosidase, invertase, ß-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH2, or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes.
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Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Estructuras Metalorgánicas/química , Metales/química , Biocatálisis , Catalasa/química , Catalasa/metabolismo , Catalasa/ultraestructura , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Enzimas Inmovilizadas/ultraestructura , Estructuras Metalorgánicas/síntesis química , Microscopía Electrónica de Rastreo , Difracción de Polvo , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/metabolismo , beta-Fructofuranosidasa/ultraestructura , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , beta-Galactosidasa/ultraestructura , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , beta-Glucosidasa/ultraestructuraRESUMEN
The role of Glu119 in S-adenosyl-L-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 10(2)-10(6) fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 10(3)-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 A and 2.75 A, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Glutamina , S-Adenosilmetionina , Alanina , Sustitución de Aminoácidos , Secuencia Conservada , Citosina , ADN/química , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/química , Agua/químicaRESUMEN
Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.