292 research outputs found
Biosynthesis and Cellular Actions of Bioactive Natural Products
The utilization of natural products as therapeutic agents has been an invaluable resource throughout medicinal history. Through the combined application of combinatorial biosynthesis, the modification of a natural product by engineered enzymes, and the isolation of new bioactive natural products, the discovery of new therapeutic agents may be achieved. This work shows the novel enzymatic interaction in the biosynthesis of a known therapeutic agent and the isolation of a new bioactive natural product.
Azinomycin A and B are antitumor natural products, isolated from Streptomyces sahachiroi. It was proposed that the biosynthesis of azinomycins is achieved through polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) machinery. In order to characterize the role of the PKS (AziB), enzymatic assays were analyzed by HPLC, LC-MS, and spectrophotometric methods. Although AziB was predicted to catalyze the production of 5-methyl-1-napthoic acid, a building block of azinomycins, a thioesterase (AziG) was found to be essential. Kinetic and crystallographic studies suggested the importance of the interaction of AziB and AziG to facilitate optimal enzymatic activity. The derivative of the AziB-AziG product is hypothesized to be assembled into the azinomycin backbone by NRPSs. In order to confirm the significance of a NRPS (AziA2) in the azinomycin biosynthetic pathway, gene knockout studies were performed. Fermentation of ΔaziA2 S. sahachiroi led to an cryptic overproduction of dimethyl furan-2,4-dicarboxylate. This showed an example of bacterial adaptation where bacteria start overproducing a new secondary metabolite to deal with the absence of a bioactive natural product.
A novel bioactive 40-membered macrolactone, Nuiapolide, was identified from a cyanobacterium collected from the Hawaiian ocean. This molecule has a rare tert-butyl side chain and nine hydroxyl groups distributed through the large hydrocarbon ring structure. Nuiapolide could inhibit metastatic activity of Jurkat at the concentration of as low as 1.3 μM without killing the cells.
In this dissertation, the new functionality of a class of enzyme, the effect of biosynthetic disruption, and a novel anti-metastatic natural product from a marine source will be discussed. This information leads to a new understanding of natural products and their biosynthesis, which will eventually help researchers developing novel therapeutic agents
A Thorough Analysis and Categorization of Bacterial Interrupted Adenylation Domains, Including Previously Unidentified Families
Interrupted adenylation (A) domains are key to the immense structural diversity seen in the nonribosomal peptide (NRP) class of natural products (NPs). Interrupted A domains are A domains that contain within them the catalytic portion of another domain, most commonly a methylation (M) domain. It has been well documented that methylation events occur with extreme specificity on either the backbone (N-) or side chain (O- or S-) of the amino acid (or amino acid-like) building blocks of NRPs. Here, through taxonomic and phylogenetic analyses as well as multiple sequence alignments, we evaluated the similarities and differences between interrupted A domains. We probed their taxonomic distribution amongst bacterial organisms, their evolutionary relatedness, and described conserved motifs of each type of M domain found to be embedded in interrupted A domains. Additionally, we categorized interrupted A domains and the M domains within them into a total of seven distinct families and six different types, respectively. The families of interrupted A domains include two new families, 6 and 7, that possess new architectures. Rather than being interrupted between the previously described a2–a3 or a8–a9 of the ten conserved A domain sequence motifs (a1–a10), family 6 contains an M domain between a6–a7, a previously unknown interruption site. Family 7 demonstrates that di-interrupted A domains exist in Nature, containing an M domain between a2–a3 as well as one between a6–a7, displaying a novel arrangement. These in-depth investigations of amino acid sequences deposited in the NCBI database highlighted the prevalence of interrupted A domains in bacterial organisms, with each family of interrupted A domains having a different taxonomic distribution. They also emphasized the importance of utilizing a broad range of bacteria for NP discovery. Categorization of the families of interrupted A domains and types of M domains allowed for a better understanding of the trends of naturally occurring interrupted A domains, which illuminated patterns and insights on how to harness them for future engineering studies
Initial recruitment and establishment of vascular plants in relation to topographical variation in microsite conditions on a recently-deglaciated moraine on Ellesmere Island, high arctic Canada
We investigated the effects of topographical positions (moraine ridge, upper side slope and lower side slope) within a recently-deglaciated young moraine on initial recruitment and establishment of vascular plants. Compared with the moraine ridge, the upper slope had similar/higher abundance of vascular plants in terms of percent cover, frequency occurrence, species number, and density/biomass of a dominating species, Salix arctica. Establishment and growth of vascular plants are generally inhibited on unstable habitats; nevertheless, on this newly-formed moraine, every attribute measured for vascular plants implied a higher probability of vascular plant recruitment on the upper slope, where substrate is less stable than on the ridge. Further, the microsite with greater vascular plant abundance, S. arctica density and S. arctica aboveground/leaf biomass accumulated more organic materials regardless of topographical positions, and such an organic accumulation was deepest on the upper slope, suggesting that relatively-successful plant establishment occurs on this site. This is further supported by the S. arctica population structure, which implies a relatively-constant juvenile supply on the upper slope. Along a slope, unstable gravels easily slide down hill. This topographical process may cause large rock size and high surface cover by rocks on the lower slope. On the upper slope, the percent cover by rocks had therefore become smaller, leading to high cover by fine-grained sediments, which retain moisture favorable for germination and growth of vascular plants. This would enhance the emergence of pioneer vascular plant species, probably resulting in higher vascular plant abundance, density and biomass of S. arctica on the upper slope. This study suggests that during primary succession following deglaciation in the high arctic the upper slope of a newly-formed glacier moraine may be an important location for the initial recruitment and establishment of pioneer vascular plant species, such as S. arctica
Unusual Substrate and Halide Versatility of Phenolic Halogenase PltM
Controlled halogenation of chemically versatile substrates is difficult to achieve. Here we describe a unique flavin-dependent halogenase, PltM, which is capable of utilizing a wide range of halides for installation on a diverse array of phenolic compounds, including FDA-approved drugs and natural products, such as terbutaline, fenoterol, resveratrol, and catechin. Crystal structures of PltM in complex with phloroglucinol and FAD in different states yield insight into substrate recognition and the FAD recycling mechanism of this halogenase
Exploiting Intermolecular Interactions between Alkyl-Functionalized Redox-Active Molecule Pairs to Enhance Interfacial Electron Transfer
The strategies to enhance electron transfer rates between redox-active, light-harvesting molecules attached to semiconductor surfaces and redox mediators in solution by modifying molecular structure are not fully investigated yet. Therefore, the design of molecules with controlled electron transfer rates remains a challenge. The aims of this work are to quantify the effect of long alkyl chain substitution on the electron transfer from cobalt(II/III) tris(2,2′-bipyridine) to organic molecules containing carbazole and thiophene and to demonstrate that alkyl chains can be used to enhance electron transfer between donor-acceptor pairs. To this end, we study the effect of using a combination of donor and acceptor molecules with and without alkyl chains on electron transfer kinetics. Using transient absorption spectroscopy, we show that when only the molecules or the mediators have long alkyl chains, electron transfer is slightly blocked as expected. Counterintuitively, electron transfer is up to 13 times faster when long alkyl chains are attached to both the redox-active molecules and the redox mediators. The faster electron transfer is explained by an alkyl-alkyl chain interaction between the donor/acceptor, leading to the proximity (trapping) of the redox mediators close to the π-conjugated backbone of the molecules. These results suggest that intermolecular interactions can be used to enhance the electron transfer rates significantly even with well-established insulating alkyl chains attached to molecules without changing the electrochemical driving force
MACULAR BLOOD FLOW CHANGES IN BRANCH RETINAL VEIN OCCLUSION EXAMINED BY OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY VARIABLE INTERSCAN TIME ANALYSIS
Purpose: To examine the relationship between changes in retinal blood flow and the recurrence of macular edema in eyes with branch retinal vein occlusion. Methods: This observational study included 32 eyes in 32 patients (18 men and 14 women) with branch retinal vein occlusion who visited the Department of Ophthalmology at Kyoto University Hospital (February 2021–November 2021). At the time of inclusion in the study, each patient underwent optical coherence tomography angiography on a macular area measuring 4 × 4 mm2. For variable interscan time analysis, different interscan times were set at 7.6 (IST7.6) and 20.6 ms (IST20.6) for the optical coherence tomography angiography. The parafoveal vessel densities were measured sectorally at IST7.6 and IST20.6, and their relationship with the longitudinal changes evident in the retinal thicknesses during the variable interscan time analysis examination and 2 months later was evaluated. Results: The parafoveal vessel densities in the affected sector was significantly greater at IST20.6 than at IST7.6 (P = 0.011). At 2 months after the variable interscan time analysis examination, 6 patients (19%) showed recurrence of macular edema involving the fovea. The difference in the parafoveal vessel densities (IST20.6 − IST7.6) in the affected sector was significantly associated with longitudinal retinal thickening in the corresponding parafovea (P = 0.020) and fovea (P = 0.014). Conclusion: In eyes with branch retinal vein occlusion, optical coherence tomography angiography variable interscan time analysis facilitated the detection of retinal blood flow changes that might be predictive for the recurrence of macular edema
Enhancement of dye regeneration kinetics in dichromophoric porphyrin-carbazole triphenylamine dyes influenced by more exposed radical cation orbitals
Reduction kinetics of oxidized dyes absorbed on semiconductor surfaces and immersed in redox active electrolytes has been mainly modeled based on the free energy difference between the oxidation potential of the dye and the redox potential of the electrolyte. Only a few mechanisms have been demonstrated to enhance the kinetics by other means. In this work, the rate constant of the reduction of oxidized porphyrin dye is enhanced by attaching non-conjugated carbazole triphenylamine moiety using iodine/triiodide and tris(2,2′-bispyridinium)cobalt II/III electrolytes. These results are obtained using transient absorption spectroscopy by selectively probing the regeneration kinetics at the porphyrin radical cation and the carbazole triphenylamine radical cation absorption wavelengths. The enhancement in the reduction kinetics is not attributed to changes in the driving force, but to the more exposed dye cation radical orbitals of the dichromophoric dye. The results are important for the development of high efficiency photo-electrochemical devices with minimalized energy loss at electron transfer interfaces
Activation of cryptic metabolite production through gene disruption: Dimethyl furan-2,4-dicarboxylate produced by Streptomyces sahachiroi
At least 65% of all small molecule drugs on the market today are natural products, however, re-isolation of previously identified and characterized compounds has become a serious impediment to the discovery of new bioactive natural products. Here, genetic knockout of an unusual non-ribosomal peptide synthetase (NRPS) C-PCP-C module, aziA2, is performed resulting in the accumulation of the secondary metabolite, dimethyl furan-2,4-dicarboxylate. The cryptic metabolite represents the first non-azinomycin related compound to be isolated and characterized from the soil bacterium, S. sahachiroi. The results from this study suggest that abolishing production of otherwise predominant natural products through genetic knockout may constitute a means to “activate” the production of novel secondary metabolites that would otherwise lay dormant within microbial genome sequences
Improved performance of porphyrin-based dye sensitised solar cells by phosphinic acid surface treatment
Chemical surface treatment of porphyrin-sensitised titania films using bis-(4-methoxyphenyl) phosphinic acid after dye adsorption, results in large improvements in DSSC efficiencies which originate primarily from higher short circuit currents. The result was attributed to a positive shift in the TiO2 quasi-Fermi level with simultaneous retardation of charge recombination. High device performances have been achieved even using simplified electrolyte matrices devoid of the common additives, LiI and t-butylpyridine
- …