Reconstitution of the Linaridin Pathway Provides Access to the Family-Determining Activity of Two Membrane-Associated Proteins in the Formation of Structurally Underestimated Cypemycin.

Cypemycin is a parent linaridin peptide known to contain nonproteinogenic dehydrobutyrine, N,N-dimethylalanine, and aminovinyl-cysteine residues. The enzymatic process by which this ribosomally synthesized peptide is formed remains elusive largely because of the deficiency of knowledge in post-translational modifications (PTMs) conducted by CypH and CypL, the two membrane-associated enzymes unique to linaridin biosynthesis. Based on heterologous reconstitution of the pathway in Streptomyces coelicolor, we report the detailed structural characterization of cypemycin as a previously unknown, d-amino acid-rich linaridin. In particular, the unprecedented family-determining activity of CypH and CypL was revealed, which, in addition to hydrolysis for removal of the N-terminal leader peptide, leads to transformation of the core peptide part of the precursor peptide through mechanistically related 16 reactions for residue epimerization (11 amino acids), dehydration (4 Thr), and dethiolation (Cys19). Subsequent functionalization for linaridin maturation includes CypD-involved aminovinyl-cysteine formation and N,N-dimethylation of the newly exposed N-terminal d-Ala residue that requires CypM activity. Genetic, chemical, biochemical, engineering, and modeling approaches were used to access the structure of cypemycin and the versatility of the CypH and CypL combination that is achieved in catalysis. This work furthers the appreciation of PTM chemistry and facilitates efforts for expanding linaridin structural diversity using synthetic biology methods.

Enzymatic Formation of an Aminovinyl Cysteine Residue in Ribosomal Peptide Natural Products.

The carboxyl-terminal (C-terminal) S-[(Z)-2-aminovinyl]-cysteine (AviCys) analogs have been identified in four families of ribosomally synthesized and post-translationally modified peptides (RiPPs): lanthipeptides, linaridins, thioamitides, and lipolanthines. Within identified biosynthetic pathways, a highly reactive enethiol intermediate, formed through an oxidative decarboxylation catalyzed by a LanD-like flavoprotein, can undergo two types of cyclization: a Michael addition with a dehydroamino acid or a coupling reaction initiated by a radical species. The collaborative actions of LanD-like proteins with diverse enzymes involved in dehydration, dethiolation or cyclization lead to the construction of structurally distinct peptide natural products with analogous C-terminal macrocyclic moieties. This concept summarizes existing knowledge regarding biosynthetic pathways of AviCys analogs to emphasize the diversity of biosynthetic mechanisms that paves the way for future genome mining explorations into diverse peptide natural products.

Characterization of a LanC-free pathway for the formation of an ll-MeLan residue and an alloAviMeCys residue in the newly identified class V lanthipeptide triantimycins.

The thioether-connected bis-amino acid lanthionine (Lan) residues are class-defining residues of lanthipeptides. Typically, the cyclization step of lanthionine formation, which relies on the addition of a cysteine to an unsaturated dehydroamino acid, is directed either by a standalone cyclase LanC (class I) or by a cyclase domain (class II-IV). However, the pathways of characterized class V members often lack a known cyclase (domain), raising a question on the mechanism by which their multi-macrocycle systems are formed. Herein, we report a new RiPP gene cluster in Streptomyces TN 58, where it encodes the biosynthesis of 3 distinct class V lanthipeptides-termed triantimycins (TAMs). TAM A1∼A3 share an N-terminal ll-MeLan residue, and only TAM A1 contains an additional internal ll-Lan residue. TAM A1 also has a C-terminal (2S, 3R)-S-((Z)-2-aminovinyl)-3-methyl-d-cysteine (alloAviMeCys) residue, which is distinct from the previously reported (2S, 3S)-AviMeCys residue in other RiPPs. Gene deletion, heterologous coexpression, and structural elucidation demonstrated that the cyclization for an ll-MeLan formation occurs spontaneously and is independent of any known lanthionine cyclase. This study provides a new paradigm for lanthionine formation and facilitates genome mining and engineering efforts on RiPPs containing (Me)Lan and (allo)Avi(Me)Cys residues.

Coupled Precipitation of Dual-Nanoprecipitates to Optimize Microstructural and Mechanical Properties of Cast Al-Cu-Mg-Mn Alloys via Modulating the Mn Contents.

The effect of Mn content on the microstructure evolution and mechanical properties of Al-Cu-Mg-x Mn alloys at ambient temperature was investigated. The findings show that in the Mn-containing alloys at the as-cast state, the blocky primary T(Al20Cu2Mn3) phase coexisting with the Al2Cu phase appeared. With the increase in Mn content, the majority of the Al2Cu phase dissolved, nd a minor amount of the T phase remained at the grain boundary after solution treatment. The rod-like TMn (Al20Cu2Mn3) nanoprecipitate was simultaneously distributed at grain boundaries and the interiors, while a high density of needle-like θ″ (Al3Cu) nanoprecipitate was also observed in the T6 state. Further increases in Mn content promoted the dispersion of the TMn phase and inhibited the growth and transformation of the θ″ phase. Tensile test results show that 0.7 wt.% Mn alloy had excellent mechanical properties at ambient temperature with ultimate tensile strength, yield strength, and fracture elongation of 498.7 MPa, 346.2 MPa, and 19.2%, respectively. The subsequent calculation of strengthening mechanisms elucidates that precipitation strengthening is the main reason for the increase in yield strength of Mn-containing alloys.

Formation of an aminovinyl-cysteine residue in thioviridamides occurs through a path independent of known lanthionine synthetase activity.

2-Aminovinyl-cysteine (AviCys) is a thioether amino acid shared by a variety of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on investigations into the biosynthesis of thioviridamide RiPPs in Streptomyces sp. NRRL S-87, we here report a path for the formation of this unusual thioether residue. This path relies on four dedicated proteins: phosphotransferase TvaCS-87, Lyase TvaDS-87, kinase homolog TvaES-87, and LanD-like flavoprotein TvaFS-87. TvaES-87 plays a critical role in effective AviCys formation. During the posttranslational modifications of the precursor peptide, it works with TvaFS-87 to form a minimum AviCys synthetase complex, which follows the combined activity of TvaCDS-87 for Thr dehydration and catalyzes Cys oxidative decarboxylation and subsequent Michael addition of the resulting enethiol nucleophile onto the newly formed dehydroamino acid residue for cyclization. With TvaES-87, TvaFS-87 activity for Cys processing can be coordinated with TvaCDS-87 activity for minimizing competitive or unexpected spontaneous reactions and forming AviCys effectively.

Numerical study of radiation effect on the municipal solid waste combustion characteristics inside an incinerator.

Due to its advantages of high degree volume reduction, relatively stable residue, and energy reclamation, incineration becomes one of the best choices for Municipal Solid Waste (MSW) disposal. However, detailed measurements of temperature and gas species inside a furnace are difficulty by conventional experimental techniques. Therefore, numerical simulation of MSW incineration in the packed bed and gas flow field was applied. In this work, a three dimensional (3-D) model of incinerator system, including flow, heat transfer, detailed chemical mechanisms, and non-gray gas models, was developed. Radiation from the furnace wall and the flame formed above the bed is of importance for drying and igniting the waste. The preheated air with high temperature is used for the MSW combustion. Under the conditions of high temperature and high pressure, MSW combustion produces a variety of radiating gases. The wavelength-depend radiative properties of flame adopted in non-gray radiation model were obtained from a statistical narrow-band model. The influence of radiative heat transfer on temperature, flow field is researched by adiabatic model (without considering radiation), gray radiation model, and non-gray radiation model. The simulation results show that taking into account the non-gray radiation is essential.

Thalamic segmentation based on improved fuzzy connectedness in structural MRI.

Thalamic segmentation serves an important function in localizing targets for deep brain stimulation (DBS). However, thalamic nuclei are still difficult to identify clearly from structural MRI. In this study, an improved algorithm based on the fuzzy connectedness framework was developed. Three-dimensional T1-weighted images in axial orientation were acquired through a 3D SPGR sequence by using a 1.5 T GE magnetic resonance scanner. Twenty-five normal images were analyzed using the proposed method, which involved adaptive fuzzy connectedness combined with confidence connectedness (AFCCC). After non-brain tissue removal and contrast enhancement, the seed point was selected manually, and confidence connectedness was used to perform an ROI update automatically. Both image intensity and local gradient were taken as image features in calculating the fuzzy affinity. Moreover, the weight of the features could be automatically adjusted. Thalamus, ventrointermedius (Vim), and subthalamic nucleus were successfully segmented. The results were evaluated with rules, such as similarity degree (SD), union overlap, and false positive. SD of thalamus segmentation reached values higher than 85%. The segmentation results were also compared with those achieved by the region growing and level set methods, respectively. Higher SD of the proposed method, especially in Vim, was achieved. The time cost using AFCCC was low, although it could achieve high accuracy. The proposed method is superior to the traditional fuzzy connectedness framework and involves reduced manual intervention in time saving.