43 research outputs found
Structural and Biochemical Characterization of the Early Steps in Fungal Indole Alkaloid Biosynthesis
Prenylated indole alkaloids are a class of natural products with great structural diversity and pharmaceutical potential. These alkaloids, which are isolated from various fungi, mostly Aspergillus and Penicillium, are often produced by homologous dimodular non-ribosomal peptide synthetase (NRPS) pathways that combine two amino acids, typically tryptophan, proline, histidine or phenylalanine, to form the alkaloid skeleton. A unique bicyclo[2.2.2]diazaoctane group is a distinctive feature of the NRPS pathway of malbrancheamide, a calmodulin inhibitor produced by Malbranchea aurantiaca. The bicyclo[2.2.2]diazaoctane is proposed to form via an intramolecular Diels-Alder reaction, but the protein that ensures stereospecificity of the reaction is unknown. This thesis describes research focused on the structural and biochemical characterization of the early steps in the malbrancheamide biosynthetic pathway, which precedes the proposed Diels-Alder reaction. In collaborative studies, I solved the first crystal structure of a fungal NRPS terminal reductase domain, PhqB R in the homologous paraherquamide pathway, which indicates that it functions as a 2-electron or 4-electron reductase. I also solved a 1.6 Ã… crystal structure of MalC, a candidate for re-oxidation of a potential 4-electron reduction product. However, the MalC structure strongly indicates that it cannot catalyze a redox reaction and its function remains to be characterized. Furthermore, MalB and MalE, two prenyltransferases in the pathway, were characterized in detail. In summary, the dissertation research provides the first structural and biochemical insights into the early steps of malbrancheamide biosynthesis and will guide protein engineering and chemoenzymatic synthesis of related compounds in the future.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/137149/1/qingydan_1.pd
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Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis.
Natural product biosynthetic pathways are replete with enzymes repurposed for new catalytic functions. In some modular polyketide synthase (PKS)Â pathways, a GCN5-related N-acetyltransferase (GNAT)-like enzyme with an additional decarboxylation function initiates biosynthesis. Here, we probe two PKS GNAT-like domains for the dual activities of S-acyl transfer from coenzyme A (CoA) to an acyl carrier protein (ACP) and decarboxylation. The GphF and CurA GNAT-like domains selectively decarboxylate substrates that yield the anticipated pathway starter units. The GphF enzyme lacks detectable acyl transfer activity, and a crystal structure with an isobutyryl-CoA product analog reveals a partially occluded acyltransfer acceptor site. Further analysis indicates that the CurA GNAT-like domain also catalyzes only decarboxylation, and the initial acyl transfer is catalyzed by an unidentified enzyme. Thus, PKS GNAT-like domains are re-classified as GNAT-like decarboxylases. Two other decarboxylases, malonyl-CoA decarboxylase and EryM, reside on distant nodes of the superfamily, illustrating the adaptability of the GNAT fold
The 2018 GaN power electronics roadmap
Gallium nitride (GaN) is a compound semiconductor that has tremendous potential to facilitate economic growth in a semiconductor industry that is silicon-based and currently faced with diminishing returns of performance versus cost of investment. At a material level, its high electric field strength and electron mobility have already shown tremendous potential for high frequency communications and photonic applications. Advances in growth on commercially viable large area substrates are now at the point where power conversion applications of GaN are at the cusp of commercialisation. The future for building on the work described here in ways driven by specific challenges emerging from entirely new markets and applications is very exciting. This collection of GaN technology developments is therefore not itself a road map but a valuable collection of global state-of-the-art GaN research that will inform the next phase of the technology as market driven requirements evolve. First generation production devices are igniting large new markets and applications that can only be achieved using the advantages of higher speed, low specific resistivity and low saturation switching transistors. Major investments are being made by industrial companies in a wide variety of markets exploring the use of the technology in new circuit topologies, packaging solutions and system architectures that are required to achieve and optimise the system advantages offered by GaN transistors. It is this momentum that will drive priorities for the next stages of device research gathered here
Drought Assessment in a Semi-Arid River Basin in China and its Sensitivity to Different Evapotranspiration Models
The Standardized Precipitation Evapotranspiration Index (SPEI) is widely used for climatological and hydrological studies, in which the estimation of potential evapotranspiration (PET) is of great importance. As many different models exist in estimating PET, the question that arises is in which way the selection of the PET model affects the calculated SPEI and the drought assessment. This study, on the basis of evaluating drought conditions over the Hexi Inland River Basin in China with long-term climate data of 18 stations by using SPEI, compared three types and eight kinds different PET models with respect to their sensitivity to the calculation of SPEI, and to drought events and drought characteristics. The results showed that the study area experienced a drying trend over the past 56 years, and the extreme drought events occurred more frequently after 2000 as a whole. All the investigated PET models were sensitive to the estimation of SPEI and to the drought assessment. When considering the alternatives of the Thornthwaite model in the calculation of SPEI for drought identification, the Blaney−Criddle equation among the temperature-based models and the Makkink equation among the radiation-based models are recommended due to the comparable results in determining the drought trends, drought events, and drought characteristics
A review on recent developments and applications of green sorbents-based solid phase extraction techniques
Sample pretreatment is an essential step in the analytical process. Solid phase extraction (SPE) procedures are widely used for clean-up and enriching analytes in sample preparation due to the complex sample matrix and low concentration level of target compounds. The sorbent is the critical part and plays a vital role in the SPE process. The combination strategies of green sample preparation (GSP) with sorbents have been taken seriously to establish high-performance and environmental-friendly SPE methods. This work mainly reviewed the capacity of different functional materials to be used as SPE sorbents which fulfilled the GSP requirements, including metal-organic frameworks (MOFs), molecularly imprinted polymers (MIPs), natural products, and carbon-based materials. Advanced computer-assisted strategies for the synthesis and usage of green SPE sorbents are also overviewed. Finally, recent advanced applications of these green adsorption materials for SPE procedure were summarized. This review aims to retrace latest research of green sorbents-based SPE techniques under the perspective of GSP, suggesting the future synthesis and application of adsorption materials should pose the features of less reagent and energy usage, less labor and time consuming, and high extraction efficiency
Quantitative 1H-NMR Method for the Determination of Tadalafil in Bulk Drugs and its Tablets
A simple, rapid, accurate, and selective quantitative nuclear magnetic resonance method for the determination of tadalafil in bulk drugs and its tablets was established and evaluated. Spectra were obtained in dimethylsulfoxide-d6 using 2,4-dinitrotoluene as the internal standard. In this study, the method’s linearity, range, limit of quantification, stability, precision, and accuracy were validated. The results were consistent with those obtained from high-performance liquid chromatography analysis. Thus, the proposed method is a useful and practical tool for the determination of tadalafil in bulk drugs and its tablets
Contamination profiles and potential health risks of environmentally persistent free radicals in PM2.5 over typical central Chinese megacity
As one of the most important transportation hubs and industrial bases in China, Zhengzhou has suffered from serious PM2.5 pollution for a long time. However, the investigation of contamination status and possible exposure risks of environmentally persistent free radicals (EPFRs) in PM2.5 from Zhengzhou is rare. In this work, a comprehensive study of pollution levels, seasonal variations, sources, and potential health risks of PM2.5-bound EPFRs in Zhengzhou was conducted for the first time. The atmospheric concentrations of EPFRs in PM2.5 from Zhengzhou ranged from 1.732 × 1012 spin m−3 to 7.182 × 1014 spin m−3 between 2019 and 2020. Relatively serious contamination was noticed in winter and spring. Primary fossil fuel combustion and Fe-mediated secondary formation were apportioned as possible sources of PM2.5-bound EPFRs in Zhengzhou. Moreover, to avert the bias of the toxicity assessment induced by utilization of incompletely extracted EPFRs from sample filter, simulatively generated EPFRs were applied to toxicological evaluations (cell viability and reactive oxygen species assays). Corresponding experimental dosages were based on the estimated adults’ annual exposure amounts of EPFRs in real PM2.5 samples. The results elucidated that EPFRs might cause growth inhibition and oxidative stress of human lung cells, suggesting the possible exposure-induced health concerns for local people in Zhengzhou. This study provides practical information of real contamination status of PM2.5-bound EPFRs in Zhengzhou, which is favorable to local air pollution control and reduction of exposure risks on public health in central China
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Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries
Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules
Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries
Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.</p
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Biofuels for a sustainable future.
Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications