Syntectonic emplacement of Late Cretaceous mafic dyke swarms in coastal southeastern China: Insights from magnetic fabrics, rock magnetism and field evidence

Magma flow directions for 6 Late Cretaceous mafic dyke swarms exposed in coastal southeastern China (SE China) were analyzed using anisotropy of magnetic susceptibility (AMS) and field evidence. Normal AMS fabrics are predominant. The AMS of the dyke swarms originates mainly from the distribution anisotropy of intersertal magnetite that crystallized during late stage magma flow or after the magma cooled. The AMS fabrics record tectonic stress combined with magma flow. Sub-vertical to vertical magma flow is inferred from symmetrical imbricated magnetic foliations of dyke walls and field evidence for 5 dyke swarms. The inferred (sub-) vertical flow directions also indicate that the magma chambers were probably just beneath the sampled locations. Low anisotropy degree, different orientations of principal AMS axes, and asymmetrical magnetic foliations of normal fabrics oblique to dyke walls indicate syntectonic emplacement of the Late Cretaceous dyke swarms under an extensional tectonic regime caused by Paleo-Pacific plate subduction.This work was financially supported by Zhejiang Provincial Natural Science Foundation of China (Grant LY12D02002). Xiaoqing Pan is further supported by international cooperation and exchange project for doctoral candidates funded by Zhejiang University (Grant 188310- 540615/001)

Insight into Structural Characteristics of Protein-Substrate Interaction in Pimaricin Thioesterase

As a polyene antibiotic of great pharmaceutical significance, pimaricin has been extensively studied to enhance its productivity and effectiveness. In our previous studies, pre-reaction state (PRS) has been validated as one of the significant conformational categories before macrocyclization, and is critical to mutual recognition and catalytic preparation in thioesterase (TE)-catalyzed systems. In our study, molecular dynamics (MD) simulations were conducted on pimaricin TE-polyketide complex and PRS, as well as pre-organization state (POS), a molecular conformation possessing a pivotal intra-molecular hydrogen bond, were detected. Conformational transition between POS and PRS was observed in one of the simulations, and POS was calculated to be energetically more stable than PRS by 4.58 kcal/mol. The structural characteristics of PRS and POS-based hydrogen-bonding, and hydrophobic interactions were uncovered, and additional simulations were carried out to rationalize the functions of several key residues (Q29, M210, and R186). Binding energies, obtained from MM/PBSA calculations, were further decomposed to residues, in order to reveal their roles in product release. Our study advanced a comprehensive understanding of pimaricin TE-catalyzed macrocyclization from the perspectives of conformational change, protein-polyketide recognition, and product release, and provided potential residues for rational modification of pimaricin TE

Theoretical Studies on the Mechanism of Thioesterase-Catalyzed Macrocyclization in Erythromycin Biosynthesis

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 <b>1</b>, and a hydrophobic pocket appropriately accommodates the substrate in the lid region, in which a pivotal prereaction state (<b>1</b>_IV′) 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

Theoretical Studies on the Catalytic Mechanism and Substrate Diversity for Macrocyclization of Pikromycin Thioesterase

Polyketide synthases (PKSs) share a subset of biosynthetic steps in construction of a polyketide, and the offload from the PKS main module of specific product release is most often catalyzed by a thioesterase (TE). In spite of the fact that various PKS systems have been discovered in polyketide biosynthesis, the molecular basis of TE-catalyzed macrocyclization remains challenging. In this study, MD simulations and QM/MM methods were combined to investigate the catalytic mechanism and substrate diversity of pikromycin (PIK) TE with two systems (PIK-TE-<b>1</b> and PIK-TE-<b>2</b>), where substrates <b>1</b> and <b>2</b> correspond to TE-catalyzed precursors of 10-deoxymethynolide and narbonolide, respectively. The results showed that, in comparison with PIK-TE-<b>2</b>, system PIK-TE-<b>1</b> exhibited a greater tendency to form a stable prereaction state, which is critical to macrocyclization. In addition, the structural characteristics of prereaction states were uncovered through analyses of hydrogen-bonding and hydrophobic interactions, which were found to play a key role in substrate recognition and product release. Furthermore, potential energy surfaces were calculated to study the molecular mechanism of macrocyclization, including the formation of tetrahedral intermediates from <i>re</i>- and <i>si</i>-face nucleophilic attacks and the release of products. The energy barrier of macrocyclization from <i>re</i>-face attack was calculated to be 16.3 kcal/mol in PIK-TE-<b>1</b>, 3.6 kcal/mol lower than that from <i>si</i>-face attack and 4.1 kcal/mol lower than that from <i>re</i>-face attack in PIK-TE-<b>2</b>. These results are in agreement with experimental observations that the yield of 10-deoxymethynolide is superior to that of narbonolide in PIK TE catalyzed macrocyclization. Our findings elucidate the catalytic mechanism of PIK TE and provide a better understanding of type I PKS TEs in protein engineering