Macrocyclic polyketides, biosynthesized by modular polyketide synthases (PKSs), have been developed successfully into generation-by-generation pharmaceuticals for numerous therapeutic areas. A great effort has been made experimentally and theoretically to elucidate the biosynthesis mechanisms, in particular for thioesterase (TE)-mediated macrocyclization, which controls the final step in the PKS biosynthesis and determines chemical structures of the final products. To obtain a better insight into the macrocyclization process (i.e., releasing step), we carried out MD simulations, QM and QM/MM calculations on complexes of 6-deoxyerythronolide B synthase (DEBS) TE and two substrates, one toward a macrocyclic product and another toward a linearly hydrolytic product. Our investigation showed the induced-fit mutual recognition between the TE enzyme and substrates: in the case of macrocyclization, a critical hydrogen-bonding network is formed between the enzyme and substrate 1, and a hydrophobic pocket appropriately accommodates the substrate in the lid region, in which a pivotal prereaction state (1IV′) with an energy barrier of 11.6 kcal/mol was captured on the potential energy surface calculation. Accompanied with the deprotonation of the prereaction state, the nucleophilic attack occurs with a calculated barrier of 9.9 kcal/mol and leads to the charged tetrahedral intermediate. Following the decomposition of the intermediate, the final macrocyclic product releases with a relatively low barrier. However, in the case of hydrolysis, such a prereaction state for cyclization was not observed in similar molecular simulations. These calculations are consistent with the previous biochemical and structural studies about the TE-mediated reactions. Our study indicated that the enzyme–substrate specificity stems from mutual molecular recognition via a prereaction state between DEBS TE and substrates, suggesting a prereaction-and-action mechanism in the TE macrocyclization and release of PKS product.Keywords: hydrolysis; macrocyclization; MD; PKS; QM/MM; thioesterase

Laccase catalyzes the oxidation of natural phenols and thereby is believed to initialize reactions in lignification and delignification. Numerous phenolic mediators have also been applied in laccase-mediator systems. However, reaction details after the primary O–H rupture of phenols remain obscure. In this work two types of isomeric phenols, EUG (eugenol) and ISO (trans-/cis-isoeugenol), were used as chemical probes to explore the enzymatic reaction pathways, with the combined methods of time-resolved UV-Vis absorption spectra, MCR-ALS, HPLC-MS, and quantum mechanical (QM) calculations. It has been found that the EUG-consuming rate is linear to its concentration, while the ISO not. Besides, an o-methoxy quinone methide intermediate, (E/Z)-4-allylidene-2-methoxycyclohexa-2,5-dienone, was evidenced in the case of EUG with the UV-Vis measurement, mass spectra and TD-DFT calculations; in contrast, an ISO-generating phenoxyl radical, a (E/Z)-2-methoxy-4-(prop-1-en-1-yl) phenoxyl radical, was identified in the case of ISO. Furthermore, QM calculations indicated that the EUG-generating phenoxyl radical (an O-centered radical) can easily transform into an allylic radical (a C-centered radical) by hydrogen atom transfer (HAT) with a calculated activation enthalpy of 5.3 kcal mol−1 and then be fast oxidized to the observed eugenol quinone methide, rather than an O-radical alkene addition with barriers above 12.8 kcal mol−1. In contrast, the ISO-generating phenoxyl radical directly undergoes a radical coupling (RC) process, with a barrier of 4.8 kcal mol−1, while the HAT isomerization between O- and C-centered radicals has a higher reaction barrier of 8.0 kcal mol−1. The electronic conjugation of the benzyl-type radical and the aromatic allylic radical leads to differentiation of the two pathways. These results imply that competitive reaction pathways exist for the nascent reactive intermediates generated in the laccase-catalyzed oxidation of natural phenols, which is important for understanding the lignin polymerization and may shed some light on the development of efficient laccase-mediator systems.

It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.

Phosphorothioation (PT), previously used in synthetic antisense drugs to arrest the transcription or translation process, is also a novel physiological modification in bacteria DNAs. In the previous study, we reported that Rp-phosphorothioation (Rp-PT) destabilizes B-type helix significantly, using a quantum-mechanics-based energy scoring function developed with a dinucleotide model (Zhang et al. J. Phys. Chem. B, 2012, 116, 10639–10648). A consequent question surfaces in the field of the phosphorothioated DNA (S-DNA) research: does the endogenous chemical modification interact with the base sequence in the bacterial genomes, e.g., in terms of the most common structure of the B-type helix? In this work, we carried out further energetic analysis on the backbone relative energies calculated with the scoring function according to 16 groups of base-step classifications. Moreover, we conducted molecular dynamics simulations of the B-helical structure with the different base-pair steps, to investigate the detailed structural changes upon the O-/S-substitution. As a result, the Rp-PT modification definitively enhances the stiffness of the backbone and differentiates backbone stability as an interaction with base-steps. Furthermore, certain exceptional sequences such as GT and CC were highlighted in the structural analysis of the sulfur local contacts and relative orientation of double strands, indicating that Rp-PT can cross-talk with particular base-steps. The special effects between the phosphorothioation and base-step may be related to the conservative consensus observed highly frequently in bacterial genomes.

S-Nitrosylation is involved in protein functional regulation and cellular signal transduction. Although intensive efforts have been made, the molecular mechanisms of S-nitrosylation have not yet been fully understood. In this work, we carried out a survey on 213 protein structures with S-nitrosylated cysteine sites and molecular dynamic simulations of hemoglobin as a case study. It was observed that the S-nitrosylated cysteines showed a lower pKa, a higher population of basic residues, a lower population of big-volume residues in the neighborhood, and relatively higher flexibility. The case study of hemoglobin showed that, compared to that in the T-state, Cysβ93 in the R-state hemoglobin possessed the above structural features, in agreement with the previous report that the R-state was more reactive in S-nitrosylation. Moreover, basic residues moved closer to the Cysβ93 in the dep-R-state hemoglobin, while big-volume residues approached the Cysβ93 in the dep-T-state. Using the four characteristics, i.e. cysteine acidity, surrounding basicity, steric hindrance, and local flexibility, a 3-dimensional model of S-nitrosylation was constructed to explain 61.9% of the S-nitrosylated and 58.1% of the non-S-nitrosylated cysteines. Our study suggests that cysteine deprotonation is a prerequisite for protein S-nitrosylation, and these characteristics might be useful in identifying specificity of protein S-nitrosylation.

Laccase is a promising oxidase with environmental applications, such as lignin degradation and chlorophenol detoxification. Laccase immobilization can significantly improve physiochemical stability and reusability compared to the free enzymes. In this work, anion effect was investigated in entrapment of Cu-alginate matrix with five types of anions, including perchlorate (ClO4−), nitrate (NO3−), sulfate (SO42−), chloride (Cl−), and acetate (CH3CO2−). Accordingly, chloride inhibition and acetate activation were detected in the o-tolidine kinetic experiments, while effects of the other three anions were much smaller. Such counteranion effects were also observed in the laccase-catalyzed biodegradation of 2,4-dichlorophenol. The results indicated that counteranions in the enzyme immobilization process are crucial for catalytic capacity, probably due to the competition with the carboxylate groups in alginate. Our results also imply that these anions might coordinate the copper cations in laccase.Counteranion is crucial for the enzyme activity of laccase immobilized with the Cu-alginate entrapment.

The mechanism of the Ru(salen)-mediated (salen = 1,2-cyclohexanediamino-N,N′-bis(3,5-di-tert-butylsalicylidene)) cyclopropanation reaction of styrene with ethyl diazoacetate (EDA) is explored with density functional theory (DFT) methods. Five proposed reaction pathways, including (1) a stepwise process containing the formation of carbene species and cyclopropanation step (path a), (2) a one-step process (path b), (3) a bis-carbene mechanism (path c), (4) a three-centered-intermediate pathway (path d), and (5) the main side reaction of dimerization of EDA (path e), are taken into consideration to determine the most favorable mechanism. Computational results indicate that path a with a barrier of 27.9 kcal/mol (trans) is superior to all other pathways. The geometries of the critical transition states are picked out for further analyses. It is found that the C–Ha group of the catalyst plays a key role in enantioselectivity. The destruction of the active center (for example, a methyl group substituent) can dramatically decrease the catalytic efficiency. In addition, the axial ligands are found to mediate energy barriers of the formation of carbene species and the cyclopropanation step in entirely opposite directions. The natural bond orbital (NBO) analyses demonstrate that carbene species show different characteristics of Fischer- or/and Schrock-type complexes. This study may help to design and develop more efficient catalysts for metal-mediated cyclopropanation reactions.

Tyrosine nitration is a covalent post-translational modification, which regulates protein functions such as hindering tyrosine phosphorylation and affecting essential signal transductions in cells. Based on up-to-date proteomics data, tyrosine nitration appears to be a highly selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. Quite a few investigations included the protein structural information from the RCSB PDB database, where near 100000 high-quality three-dimensional structures are available. In this work, we analyzed the local protein structures and amino acid topological networks of the nitrated and non-nitrated tyrosine sites in nitrated proteins, including neighboring atomic distribution, amino acid pair (AAP) and amino acid triangle (AAT). It has been found that aromatic and aliphatic residues, particularly with large volume, aromatic, aliphatic, or acidic side chains, are disfavored for the nitration. After integrating these structural features and topological network features with traditional sequence features, the predictive model achieves a sensitivity of 63.30% and a specificity of 92.24%, resulting in a much better accuracy compared to the previous models with only protein sequence information. Our investigation implies that the site selectivity may stem from a more open, hydrophilic and high-pH chemical environment around the tyrosine residue.

A dilemma about whether thionitroxide radical (RSNHO) or S-nitrosothiol (RSNO) is observed in protein S-nitrosylation has arisen recently. To illustrate the effect of chemical environment on these structures, this paper presents quantum mechanical molecular dynamics of thionitroxide, and cis- and trans-S-nitrosothiols in the gas phase, methanol, and water. By using Car-Parrinello molecular dynamics (CPMD), we have observed that there is free rotation about the S-N bond at 300 K in thionitroxide, but no such rotation is observed for S-nitrosothiol. The C-S-N-O torsion angle distribution in thionitroxide is significantly dependent upon the surrounding environment, leading to either gauche-, cis-, or trans-conformation. In the case of S-nitrosothiol the C-S-N-O plane is twisted slightly by 5°–15° in the cis-isomer, while the periplanar structure is well-retained in the trans-isomer. The calculated results are in agreement with the X-ray crystallographic data of small molecular RSNO species. Interestingly, for both compounds, the CPMD simulations show that solvation can cause a decrease in the S-N bond length. Moreover, the oxygen atom of thionitroxide is found to be a good hydrogen-bond acceptor, forming an oxyanionhole-like hydrogen bonding network.

Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bacterial DNA electrophoresis. To understand structural characteristics of the thio-substituted DNA, we have investigated the correlation between the relative energy of phosphate/phosphorothioate linkage and the backbone torsions. The relative energies (R.E.) computed by the quantum mechanical method, the PBE1PBE(CPCM, solvent=water)//PBE1PBE/6-31+G(2df) level of theory, were used to construct energy-scoring functions against backbone torsion variables, resulting in the squared correlation coefficients r2 of 0.90–0.95. Then, the DNA energy alteration by phosphorothioation is estimated with the relative energy difference (ΔR.E.) between phosphate and phosphorothioate of the phosphate linkages in the DNA crystallographic database (NDB). As a result, Rp-phosphorothioation shifts the relative energy of B-helical structures by 2.7 ± 3.4 kcal/mol, destabilizing about 95% linkages, while Sp-phosphorothioation by −1.4 ± 2.4 kcal/mol, stabilizing over 84% linkages in the data sets. The B-helical destabilization is likely caused by the steric effect between the sulfur atom of Rp-phosphorothioate and the neighboring C–H groups of deoxyribose on the groove wall in B-helix. The unfavorable interaction may be magnified by the increasing rigidness of P–O-involving backbone torsions α and ζ upon the nonbridging phosphorothioations. Since B-helix is the most prevalent DNA double-helical structure and Rp-phosphorothioation is the exclusive configuration in bacteria thio-DNA found to date, the observed stereospecificity-destabilization correlation may reflect a structure–function relationship of biological DNA-phosphorothiation.

It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.

Laccase catalyzes the oxidation of natural phenols and thereby is believed to initialize reactions in lignification and delignification. Numerous phenolic mediators have also been applied in laccase-mediator systems. However, reaction details after the primary O–H rupture of phenols remain obscure. In this work two types of isomeric phenols, EUG (eugenol) and ISO (trans-/cis-isoeugenol), were used as chemical probes to explore the enzymatic reaction pathways, with the combined methods of time-resolved UV-Vis absorption spectra, MCR-ALS, HPLC-MS, and quantum mechanical (QM) calculations. It has been found that the EUG-consuming rate is linear to its concentration, while the ISO not. Besides, an o-methoxy quinone methide intermediate, (E/Z)-4-allylidene-2-methoxycyclohexa-2,5-dienone, was evidenced in the case of EUG with the UV-Vis measurement, mass spectra and TD-DFT calculations; in contrast, an ISO-generating phenoxyl radical, a (E/Z)-2-methoxy-4-(prop-1-en-1-yl) phenoxyl radical, was identified in the case of ISO. Furthermore, QM calculations indicated that the EUG-generating phenoxyl radical (an O-centered radical) can easily transform into an allylic radical (a C-centered radical) by hydrogen atom transfer (HAT) with a calculated activation enthalpy of 5.3 kcal mol−1 and then be fast oxidized to the observed eugenol quinone methide, rather than an O-radical alkene addition with barriers above 12.8 kcal mol−1. In contrast, the ISO-generating phenoxyl radical directly undergoes a radical coupling (RC) process, with a barrier of 4.8 kcal mol−1, while the HAT isomerization between O- and C-centered radicals has a higher reaction barrier of 8.0 kcal mol−1. The electronic conjugation of the benzyl-type radical and the aromatic allylic radical leads to differentiation of the two pathways. These results imply that competitive reaction pathways exist for the nascent reactive intermediates generated in the laccase-catalyzed oxidation of natural phenols, which is important for understanding the lignin polymerization and may shed some light on the development of efficient laccase-mediator systems.