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Metal-organic framework (MOF) membranes have enormous potential in separation applications. There are several MOF membranes grown on polymer substrates aimed for scale-up, but their brittleness hampers any industrial application. Herein, intergrown continuous polypropylene (PP)-supported ZIF-8 membranes have been successfully synthesized via fast current-driven synthesis (FCDS) within 1 h. The PP-supported ZIF-8 membranes exhibit a promising separation factor of 122 ± 13 for binary C3H6-C3H8 mixtures combined with excellent flexibility behavior. The C3H6/C3H8 separation performance of the PP-supported ZIF-8 membrane was found to be constant after bending the supported ZIF-8 film with a curvature of 92 m-1. This outstanding mechanical property is crucial for practical applications. Moreover, we further synthesized ZIF-8 membranes on various polymer substrates and even polymer hollow fibers to demonstrate the production scalability.

Surface and interfacial behavior of protein molecules are crucial for the protein function involved in many biochemical processes and biomedical products such as enzyme design, bio-separation, drug design and delivery. This article is devoted to an overview of design and regulation of the surface and interfacial behavior of protein molecules. The improvement of enzyme surface such as the directed evolution and the rational design of enzymes is introduced at first, followed by the rational design of protein interface for the protein assembly. Thereafter, the design of micro-environment and ligands are described as two examples for the design guided by protein surface. Then the design of protein surface and interface with the help of artificial intelligence will be discussed.

NOV1 is a stilbene cleavage oxygenase (SCO) responsible for the oxidative cleavage of the central double bond of stilbenes, forming two phenolic aldehydes. In the active site of NOV1, the iron cofactor is coordinated by four histidines and no negatively charged ligand, which is unusual for iron-containing enzymes. Here, on the basis of the recently obtained crystal structure of NOV1, we performed quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the reaction mechanism of NOV1. According to our calculations, two binding modes of O2 to Fe(II) (end-on and side-on modes) have been recognized, and the end-on mode is suitable for catalysis. The side-on mode, which has been obtained from the crystal, is an inactive form, which should first change to the end-on mode to initiate the catalytic reaction. For the reactant in end-on mode, the triplet and quintet states correspond to very similar energies. Of the three considered pathways, two pathways (path_a and path_b1, which differ only in the order of the first C–O bond formation) involving the dioxetane intermediate are calculated to be favorable, which can confirm the dioxygenase mechanism of NOV1. However, the pathway (path_b2) involving the epoxide intermediate corresponds to high energy barriers for the formation of the second C–O bond (27.49 and 31.56 kcal mol−1 on the triplet and quintet state surfaces) and can be ruled out. However, in path_b2, the formation of the epoxide intermediate is easy, which may react with the solvent water (non-enzymatic reaction) to form the by-product. In addition, the different protonation states of 4′-OH of the substrate and K134, as well as the coordination of additional water molecules with the iron, do not influence the reaction barrier.

International audience; Nucleoside analogues represent an important class of drug candidates. With the aim of searching for novel bioactive nucleosides, we developed an efficient synthetic way to construct a series of aryl 1,2,3-triazole acyclic C-azanucleosides via Huisgen 1,3-dipolar cycloaddition. The aryl 1,2,3-triazole motifs within these azanucleosides showed coplanar features, suggesting they could act as surrogates for large planar aromatic systems or nucleobases. Moreover, several aryltriazole acyclic C-azanucleosides bearing long alkyl chains exhibited potent antiproliferative activity against various cancer cell lines via induction of apoptosis. Most interestingly, the lead compound significantly down-regulated the key proteins involved in the heat shock response pathway, representing the first anticancer acyclic azanucleoside with such a mode of action. These novel aryl 1,2,3-triazole cyclic C-azanucleosides therefore serve as promising paradigms for further exploring anticancer drug candidates.

Posttranslational modification is a critical process in the cellular regulation, an example of which is the carboxylation of lysine. Pyruvate carboxylase is an enzyme, in which a carboxylated lysine has been found in the metal coordination shell of the active center. In our previous study, the reaction mechanism of the carboxyl transferase domain of the pyruvate carboxylase from Staphylococcus aureus has been investigated by using QM/MM calculations. The suggested mechanism supports the previous proposal, and most of the results are consistent with the experimental data. However, the calculated overall barrier is too high for an enzymatic reaction, which may be the result of the used model failing to take into account the modification of the metal-coordinated lysine. Here, we present a successive study to investigate the importance of lysine carboxylation in the reaction and also to examine if the Zn-coordinated water molecule is required for the catalysis. The reaction mechanism from the new models is consistent with the previous suggestion. More importantly, the energy barriers of all elementary steps are calculated to be much lower than those in our previous work. Notably, the calculated barrier of the overall reaction is ~ 14 kcal/mol, which is in good agreement with the experimental value. Therefore, this study supplies a theoretical evidence for the importance of the modification of Zn-coordinated lysine in the pyruvate carboxylase-catalyzed reaction. In addition, the water molecule in the zinc coordination shell is suggested to contribute to the catalysis.

Bioproduction of organic acids under low pH condition without adding inducer and neutralizer is an economical craft to decrease downstream cost. Candida glycerinogenes had higher tolerances to low pH and lactic acid than Saccharomyces cerevisiae. QRT-PCR analysis showed that four of fifteen candidates functioned as potentially acid-inducible promoters in C. glycerinogenes. In particular, PCggmt1 showed the strongest induction ability at pH 2.5. Fluorescence analysis indicated that the induction ability of PCggmt1 gradually increased as the pH decreased. In addition, PCggmt1 had the binding sites of Msn2p/4p, Azf1p, and Nrg1p. PCggmt1 was further used to control the expression of lactate dehydrogenase gene from Rhizopus oryzae in C. glycerinogenes. Compared with pH 5.5, the specific activity of lactate dehydrogenase and lactic acid titer at pH 2.5 were increased by 229% and 218%, reaching 13.8 mU/mg and 12.3 g/L, respectively. These results presented here showed a potential to produce organic acid economically at low pH by the stress tolerant C. glycerinogenes and the novel low-pH inducible promoter PCggmt1.

Salicylate hydroxylase (SALH) is a Flavin-dependent monooxygenase responsible for the transformation of salicylate to catechol. In this article, on the basis of the crystal structure obtained from Pseudomonas putida S-1, we performed combined quantum mechanical/molecular mechanical (QM/MM) calculations to investigate the reaction mechanism of SALH. Since the formation of C4a-hydroperoxyflavin has been theoretically proven to be a barrierless process, our calculations started from the C4a-hydroperoxyflavin intermediate. The whole enzymatic reaction contains two parts: the hydroxylation and decarboxylation. Our calculation results indicate that the deprotonated substrate is the active form, whereas the neutral form of salicylate corresponds to very a high energy barrier (39.8 kcal/mol) for the hydroxylation process, which is in line with the experimental result that the optimum pH is 7.6. The calculated results with the deprotonated substrate indicate that the hydroxylation and decarboxylation occur in a stepwise manner and the decarboxylation process is calculated to be the rate-limiting step with an energy barrier of 14.5 kal/mol. Calculations using different functionals (B3LYP, BP, BVWN, PBE, M06 and TPSSH) suggest that the catalytic reaction is highly exothermic, which is consistent with its similar enzyme (PHBH).

ZIF-8 membranes with inborn-suppressed linker mobility sharpen molecular sieving with C3H6/C3H8 separation factor above 300., Metal-organic framework (MOF) membranes show great promise for propene/propane separation, yet a sharp molecular sieving has not been achieved due to their inherent linker mobility. Here, zeolitic imidazolate framework ZIF-8–type membranes with suppressed linker mobility are prepared by a fast current–driven synthesis (FCDS) strategy within 20 min, showing sharpened molecular sieving for propene/propane separation with a separation factor above 300. During membrane synthesis, the direct current promotes the metal ions and ligands to assemble into inborn-distorted and stiffer frameworks with ZIF-8_Cm (a newly discovered polymorph of ZIF-8) accounting for 60 to 70% of the membrane composition. Molecular dynamics simulations further verify that ZIF-8_Cm is superior to ZIF-8_I 4¯3m (the common cubic phase) for propene/propane separation. FCDS holds great potential to produce high-quality, ultrathin MOF membranes on a large scale.

Serum paraoxonase 1 (PON1) is a calcium-dependent enzyme that can catalyze the hydrolysis of multiple substrates, including lactones, thiolactones, carbonates, esters and phosphotriesters, as well as the formation of a variety of lactones. To better understand the lactonase mechanism of PON1, the hydrolysis of dihydrocoumarin, which is considered as a native substrate of PON1, has been investigated by using a combined quantum mechanics and molecular mechanics (QM/MM) approach. Two possible reaction pathways with either Glu53 or His115 acts as the general base have been considered. On the basis of our calculations, these two pathways correspond to the overall energy barriers of 12.5 and 9.0 kcal mol−1, respectively. During the catalytic reaction, if one of the two residues (Glu53 and His115) acts as the catalytic base, the other one forms strong hydrogen bonding interaction with the attacking hydroxide to facilitate the hydrolysis. However, mutation studies reveal that Glu53 is necessary for hydrolysis, whereas His115 is not essential but can promote the activity of PON1. Natural population analysis indicates that the catalytic Ca2+ does not act as Lewis acid but plays structural role in fixing the orientations of the substrate and related residues. In addition, Asp269 is found to coordinate with Ca2+ cation and facilitate the protonation of the alkoxide leaving group by forming hydrogen bond with lactone. These results can explain the fact that mutation of Glu53 results in the loss of activity of PON1, and the hydrolysis of dihydrocoumarin is unaffected by mutation of H115.

Benzaldehyde lyase (BAL) is a versatile thiamin diphosphate (THDP)-dependent enzyme with widespread synthetic applications in industry. Besides lyase activity, BAL also performs the functions as carboligase and decarboxylase. Unlike many other THDP-dependent enzymes, the active center of BAL is devoid of any acid-base amino acid residues except Glu50 and His29, and therefore, the catalytic mechanism of BAL is unusual. In this article, the dissociation mechanism of (R)-benzoin to benzaldehyde catalyzed by BAL has been studied by using density functional theory method. The calculation results indicate that the whole reaction consists of four elementary steps, and at least two steps contribute to rate-limiting. A big difference with other THDP-dependent enzymes is that, in the first stage of the reaction, the ligation of substrate and THDP ylide is not companied by proton transfer, and in the subsequent transition states and intermediates, the carbonyl oxygen always exists in the form of anion. Gln113, His29, and 4-amino group of THDP are found to have the function to stabilize the transition states and intermediates. His29 acts as the proton acceptor in step 2 and proton donor in step 3 using one water molecule as mediator. (c) 2013 Wiley Periodicals, Inc.

The Taxus canadensis phenylalanine aminomutase (TcPAM) catalyze the isomerization of (S)-alpha-phenylalanine to the (R)-beta-isomer. The active site of TcPAM contains the signature 5-methylene-3,5-dihydroimidazol-4-one (MIO) prosthesis, observed in the ammonia lyase class of enzymes. Up to now, there are two plausible mechanisms for these MIO-dependent enzymes, i.e., the amino-MIO adduct mechanism and the Friedel-Crafts-type reaction mechanism. In response to this mechanistic uncertainty, the phenylalanine aminomutase mechanism was investigated by using density functional methods. The calculation results indicate that: (1) the reaction prefers the amino-MIO adduct mechanism where the 2,3-amine shift process contains six elementary steps; (2) the ammonia elimination step proceeds through an E2 mechanism; (3) a single C1-C alpha bond rotation of 180 degrees in the cinnamate skeleton occurs in the active site prior to the rebinding of NH2 group to the cinnamate. This can be used to explain the stereochemistry of the TcPAM reaction product which is contrary to those of the PaPAM and SgTAM enzymes. Based on these calculations, the roles of important residues in the active site were also elucidated. (C) 2013 Elsevier Inc. All rights reserved.

It is proposed that the catalysis of GH1 enzymes follows a double-displacement mechanism involving a glycosylation and a deglycosylation steps. In this article, the deglycosylation step was studied using quantum mechanical/molecular mechanical (QM/MM) approach. The calculation results reveal that the nucleophilic water (Wat1) attacks to the anomeric C1, and the deglycosylation step experiences a barrier of 21.4 kcal/mol from the glycosyl-enzyme intermediate to the hydrolysis product, in which an oxocarbenium cation-like transition state (TS) is formed. At the TS, the covalent glycosyl-enzyme bond is almost broken (distance of 2.45 angstrom), and the new covalent bond between the attacking oxygen of the water molecule and C1 is basically established (length of 2.14 angstrom). In addition, a short hydrogen bridge is observed between the nucleophilic E386 and the C2OH of sugar ring (distance of 1.94 angstrom) at the TS, which facilitates the ring changing from a chair form to half-chair form, and stabilizes the oxocarbenium cation-like TS. (c) 2013 Wiley Periodicals, Inc.

Epinephrine is a naturally occurring adrenomedullary hormone that transduces environmental stressors into cardiovascular actions. As the only route in the catecholamine biosynthetic pathway, Phenylethanolamine N-methyltransferase (PNMT) catalyzes the synthesis of epinephrine. To elucidate the detailed mechanism of enzymatic catalysis of PNMT, combined quantum-mechanical/molecular-mechanical (QM/MM) calculations were performed. The calculation results reveal that this catalysis contains three elementary steps: the deprotonation of protonated norepinphrine, the methyl transferring step and deprotonation of the methylated norepinphrine. The methyl transferring step was proved to be the rate-determining step undergoing a SN2 mechanism with an energy barrier of 16.4 kcal/mol. During the whole catalysis, two glutamic acids Glu185 and Glu219 were proved to be loaded with different effects according to the calculations results of the mutants. These calculation results can be used to explain the experimental observations and make a good complementarity for the previous QM study. (C) 2012 Elsevier B.V. All rights reserved.

The quantum-mechanical/molecular-mechanical (QM/MM) method was used to study the glycosylation mechanism of rice BGlu1 β-glucosidase in complex with laminaribiose. The calculation results reveal that the glycosylation step experiences a concerted process from the reactant to the glycosyl-enzyme complex with an activation barrier of 15.7 kcal/mol, in which an oxocarbenium cation-like transition state (TS) is formed. At the TS, the terminal saccharide residue planarizes toward the half-chair conformation, and the glycosidic bond cleavage is promoted by the attacks of proton donor (E176) on glycosidic oxygen and nucleophilic residue (E386) on the anomeric carbon of laminaribiose. Both the nucleophilic glutamate (E386) and acid/base catalyst (E176) establish shorter hydrogen bridges with the C₂-hydroxyl groups of sugar ring, which play an important role in the catalytic reaction of rice BGlu1 β-glucosidase.

Recent theoretical studies on N-heterocyclic carbenes catalyzed Staudinger reactions revealed that the stereoselectivities are determined by an elementary reaction step, that is, the reaction of an imine with N-heterocyclic carbene-ketene zwitterionic intermediate ( INT N-k ). To better understand the stereoselectivities of some of the experimentally reported reaction systems, the corresponding transition states were studied further by using an ONIOM approach. A combination of B3LYP/6-31G(d) and PM3 levels of theory was used for transition state optimization, and M06-2X/6-31+G(d,p) level for single point calculations. The calculations were found to provide predictions that were in qualitative agreement with the sense and magnitude of experimental stereoselectivities. By analyzing the electronic structures of the transition states, the origin of divergent stereoselectivities in different N-heterocyclic carbenes catalyzed system can be attributed to the cooperation of the electrostatic and steric effects involved in the reaction of imines with N-heterocyclic carbene-ketene intermediates. These results may provide useful information for selecting the N-heterocyclic carbene catalysts in the synthesis of β-lactams.

The structural rearrangement of the ligand binding domain (LBD) of human Vitamin D receptor (hVDR) complexed with 1α, 25-dihydroxyvitamin D3 (natural ligand) and its analogues (denoted as b and c) was studied by molecular dynamics (MD) simulations. MD simulations revealed that these ligands could induce different structural changes of LBD, in which 1α, 25-dihydroxyvitamin D3 only led to a minute change, suggesting that LBD adopted its canonical active conformation upon binding the natural ligand, while b and c could provoke a clear structural rearrangement of the LBD. In complex of hVDR-LBD/b, it is found that helix 6 (H6) and subsequent loop 6-7 shift outward and the last turn of H11 shifts away from H12, which generate a new cavity at the bottom of binding pocket to accommodate the extra butyl group on the side chain of ligand b. As for hVDR-LBD/c, the steric exclusion of the second side chain of ligand c makes the N-terminal of H7 move outsides and C-terminal of H11 close to H12, expanding the bottom of the pocket. These calculation results agree well the experimental observations. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

Archaeal fructose-1,6-bisphosphate aldolase/phosphatase (FBPA/P) is a newly identified unusual bifunctional enzyme (Nature, 2010, 464, 1077), which contains one single catalytic domain but catalyzes two chemically distinct reactions of gluconeogenesis. It is different from the ordinary enzymes whose active sites are responsible for a specific reaction. To explore the catalytic characteristic of FBPA/P, the aldol condensation mechanism of bifunctional FBPA/P has been investigated using quantum mechanics/molecular mechanics (QM/MM) method. The whole reaction process can be divided into two half-reactions involving seven elementary steps. A Schiff base intermediate is theoretically confirmed, agreeing well with the recently resolved crystal structures (Nature, 2011, 478, 538). The free energy barrier of the rate-limiting step is calculated to be 22.2 kcal mol(-1), which is a concerted process of a nucleophilic attack by the enolic carbon to the ketonic carbon and a proton transfer from Tyr229 to the ketonic oxygen. Lys232 plays an important role in forming a Schiff base intermediate with the substrate (DHAP). Tyr229 functions as a proton shuttle during the catalysis. This is the first theoretical study on the aldol condensation mechanism of FBPA/P, which may provide useful information for understanding bifunctional enzymes.

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme responsible for the degradation of ADP-ribose polymers. Very recently, the first crystal structure of PARG was reported (Dea Slade, et al., Nature 477 (2011) 616), and a possible SN1-type-like mechanism was proposed. In this work, we present a computational study on the hydrolysis of glycosidic ribose–ribose bond catalyzed by PARG using hybrid density functional theory (DFT) methods. Based on the crystal structure of PARG, three models of the active site were constructed. The calculation results suggest that the degradation of poly(ADP-ribose) follows an SN2 mechanism, and the oxocarbenium expected by Dea Slade is a possible transition state but not an intermediate. The calculated reaction pathway agrees with the proposed mechanism. According to the computational models with different sizes, the roles of key residues are elucidated. Our results may provide useful information for the subsequent experimental and theoretical studies on the structure and functional relationships of PARG.

Pyruvate decarboxylase (PDC) is a typical thiamin diphosphate (ThDP)-dependent enzyme with widespread applications in industry. Though studies regarding the reaction mechanism of PDC have been reported, they are mainly focused on the formation of ThDP ylide and some elementary steps in the catalytic cycle, studies about the whole catalytic cycle of PDC are still not completed. In these previous studies, a major controversy is whether the key active residues (Glu473, Glu50′, Asp27′, His113′, His114′) are protonated or ionized during the reaction. To explore the catalytic mechanism and the role of key residues in the active site, three whole-enzyme models were considered, and the combined QM/MM calculations on the nonoxidative decarboxylation of pyruvate to acetaldehyde catalyzed by PDC were performed. According to our computational results, the fundamental reaction pathways, the complete energy profiles of the whole catalytic cycle, and the specific role of key residues in the common steps were obtained. It is also found that the same residue with different protonation states will lead to different reaction pathways and energy profiles. The mechanism derived from the model in which the residues (Glu473, Glu50′, Asp27′, His113′, His114′) are in their protonated states is most consistent with experimental observations. Therefore, extreme care must be taken when assigning the protonation states in the mechanism study. Because the experimental determination of protonation state is currently difficult, the combined QM/MM method provides an indirect means for determining the active-site protonation state.

Receptor-based comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on a series of 54 progesterone receptor (PR) inhibitors. The established CoMFA model from the training set gives statistically significant results with the cross-validated q (2) of 0.534 and non-cross-validated [Formula: see text] of 0.947. The best CoMSIA model was derived by the combination of steric field and hydrophobic field with a q (2) of 0.615 and [Formula: see text] of 0.954. A test set of 18 compounds was used to validate the predictive ability of the two models. The predicted correlation coefficients [Formula: see text] are 0.681 and 0.677 for CoMFA and CoMSIA models, respectively. Based on the CoMFA maps, the key structural characters of progesterone receptor inhibitors are identified. Moreover, the binding modes of oxindoles and benzimidazol-2-ones are also given by the quantum mechanical/molecular mechanical (QM/MM) calculations. This may provide useful information for drug design.

Limonene 1,2-epoxide hydrolase (LEH) is completely different from those of classic epoxide hydrolases (EHs) which catalyze the hydrolysis of epoxides to vicinal diols. A novel concerted general acid catalysis step involving the Asp101–Arg99–Asp132 triad is proposed to play an important role in the mechanism. Combined quantum-mechanical/molecular-mechanical (QM/MM) calculations gave activation barriers of 16.9 and 25.1 kcal/mol at the B3LYP/6-31G(d,p)//CHARMM level for nucleophilic attack on the more and less substituted epoxide carbons, respectively. Furthermore, the important roles of residues Arg99, Tyr53 and Asn55 on mutated LEH were evaluated by QM/MM-scanned energy mapping. These results may provide an explanation for site-directed mutagenesis.

Combined quantum-mechanical/molecular-mechanical (QM/MM) approaches have been applied to investigate the detailed reaction mechanism of human O(6)-alkylguanine-DNA alkyltransferase (AGT). AGT is a direct DNA repair protein that is capable of repairing alkylated DNA by transferring the methyl group to the thiol group of a cysteine residue (Cys145) in the active site in an irreversible and stoichiometric reaction. Our QM/MM calculations reveal that the methyl group transferring step is expected to occur through two steps, in which the methyl carbocation generating step is the rate-determining step with an energy barrier of 14.4 kcal/mol at the QM/MM B3LYP/6-31G(d,p)//CHARMM22 level of theory. It is different from the previous theoretical studies based on QM calculations by using a cluster model in which the methyl group transferring step is a one-step process with a higher energy barrier.

The ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) for 82 inhibitors of 25-dehydro-1α-hydroxyvitamin D3 -26,23-lactone analogs has been studied by using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models. The established CoMFA model in training set gives a cross-validated q(2) value of 0.516 and a non-cross-validated rncv (2) value of 0.667, while the CoMSIA model results in q(2) =0.517 and rncv (2) =0.632. In general, the predictive ability of the CoMFA model is superior to that of the CoMSIA model, with rpred (2) =0.639 for the CoMFA and rpred (2) =0.619 for the CoMSIA model. Based on the CoMFA contour maps, some key structural characters of vitamin D3 analogs responsible for inhibitory activity are identified, and some new C2α-modified 24-alkylvitamin D3 lactone analogs with high predicted pIC50 values are designed. The ligand functional group mutations by FEP simulation and docking studies reveal the rationality of the molecular design.