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41.
Jeaphianne P. M. van Rijn Andrés M. Escorcia Walter Thiel 《Journal of computational chemistry》2019,40(21):1902-1910
Combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the reaction mechanism of taxadiene synthase (TXS). TXS catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to taxadiene (T) and four minor cyclic products. All these products originate from the deprotonation of carbocation intermediates. The reaction profiles for the conversion of GGPP to T as well as to minor products were calculated for different configurations of relevant TXS carbocation complexes. The QM region was treated at the M06-2X/TZVP level, while the CHARMM27 force field was used to describe the MM region. The QM/MM calculations suggest a reaction pathway for the conversion of GGPP to T, which slightly differs from previous proposals regarding the number of reaction steps and the conformation of the carbocations. The QM/MM results also indicate that the formation of minor products via water-assisted deprotonation of the carbocations is highly exothermic, by about −7 to −23 kcal/mol. Curiously, however, the computed barriers and reaction energies indicate that the formation of some of the minor products is more facile than the formation of T. Thus, the present QM/MM calculations provide detailed insights into possible reaction pathways and into the origin of the promiscuity of TXS, but they do not reproduce the product distribution observed experimentally. © 2019 Wiley Periodicals, Inc. 相似文献
42.
The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (ArgII) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting ArgII with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against ArgII. These results might be associated with increased L-arginine bioavailability. Collectively, these results suggest that ArgII may be a valuable target in age-dependent vascular diseases. 相似文献
43.
Guifa Zhai Wenyan Wang Dr. Wei Xu Guo Sun Chaoqun Hu Xiangming Wu Zisong Cong Liang Deng Yanrong Shi Prof. Dr. Peter F. Leadlay Prof. Dr. Heng Song Prof. Dr. Kui Hong Prof. Dr. Zixin Deng Prof. Dr. Yuhui Sun 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(50):22926-22930
The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis. 相似文献
44.
设计合成了新的甲氨蝶呤(methotrexate, MTX)衍生物1~4, 在这些新化合物中, 将MTX分子中10-位对氨基苯甲酰谷氨酸砌块移植到4-位, 同时在6-位引入苯环芳香基以及甲基等基团. 生物活性测试结果显示, 化合物1~4具有与MTX相似的抑制iNOS活性的作用; 相对于MTX, 选测的化合物2和4明显地增强了抑制K-562白血病细胞株生长的活性. 本研究为进行MTX的结构修饰开辟了新途径, 2-氨基-4-[N-(对氨基苯甲酰谷氨酸)-基]-6-取代基蝶啶衍生物可成为潜在的抗肿瘤候选药物被进一步研究. 相似文献
45.
Yujie Wang Lifang Ruan Wai-Hung Lo Hong Chua Hoi-Fu Yu 《Applied biochemistry and biotechnology》2006,132(1-3):1015-1022
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon
and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant
plasmids, pBE2C1, and pBE2C1AB, containing one or two PHA synthse, genes, respectively. The two plasmids were inserted into
Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example
of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxyde-canoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. 相似文献
46.
Although there are a number of existing assays for monitoring the activity of both isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), none have demonstrated the qualities required for screening a mutant library. Hence, enzyme-linked immunosorbent assays (ELISAs) for IPNS and DAOCS were developed based on the detection of the catalytic turnover products isopenicillin N and cephalexin/phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), respectively. These assays are relatively fast compared to existing assays, such as the hole-plate bioassay, and are amenable with high-throughput screening. Both the IPNS and DAOCS-ELISAs were optimised for use with crude protein extracts rather than purified protein, thereby eliminating any additional time required for purification. The ELISA developed for the detection of cephalexin had an IC50 value of 154 ± 9 ng mL−1 and LOD of 7.2 ± 2.2 ng mL−1 under conditions required for the assay. Good recoveries and correlation was observed for spiked samples when the concentration of crude protein was kept below 1 mg mL−1. The DAOCS-ELISA was found to have increased sensitivity compared to the hole-plate bioassay (10.3 μg mL−1). The IPNS-ELISA did not significantly increase the sensitivity (approximately 5 μg mL−1) compared to that of the hole-plate bioassay (16 μg mL−1) for isopenicillin N. The minimum amount of crude protein extract required for producing detectable amounts of product for both assays was below 0.5% of the maximum amount of protein that the assay could contain without any effect on the ELISA. This suggests that when screening a mutant library, mutants producing low amounts of the product could still be detected using these assays. 相似文献
47.
The selectivity of substrate in substrate–enzyme complexation of heptaprenyl diphosphate synthase was directly investigated
using colloidal probe atomic force microscopy (AFM). This enzyme is composed of two dissociable subunits, which exhibits a
catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). We have recently succeeded to directly demonstrate a specific interaction involved
in this enzyme reaction and obtain new insights into the molecular mechanism of the reaction using the approach based on the
colloidal probe AFM. The AFM measurement showed the adhesive force between the subunits only in the presence of both Mg2+ and FPP. In this study, we studied the substrate selectivity in the complexation by monitoring the adhesive force. The substrates
studied are pyrophosphate (PPi), isopentenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl monophosphate (FP), and
farnesyl geranyl diphosphate (FGPP). No adhesion was observed in the case of PPi, IPP, and GPP. On the other hand, the significant
adhesion was observed for phosphate derivatives, which bear prenyl units longer than three. This is in good agreement with
the selectivity of the substrates by this enzyme, which catalyzes the condensation reaction of four IPP molecules with FPP
to give heptaprenyl (C35) diphosphates. Our study showed a useful methodology for examining the elemental processes of biological reactions. 相似文献
48.
Jiao YL Wang LH Dong XY Chen YF Zong Y Gao Y Ren N Guo AY Zhang XQ Jiao BH 《Applied biochemistry and biotechnology》2008,149(1):67-78
Using the consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction method, 26 new ketoacyl synthase (KS)
fragments were isolated from a marine sediment sample in the East China Sea (ECS) and analyzed by construction of a phylogenetic
tree. With a digoxigenin-labeled KS gene fragment used as a probe, a partial polyketide synthase (PKS) gene cluster was isolated
and identified by hybridization screening of a marine sediment sample metagenome fosmid library constructed for this study.
A new acyltransferase (AT) gene was cloned from the PKS gene cluster and heterogeneously expressed as a protein fused to maltose-binding
protein (MBP). Ultraviolet spectrophotometry was used to study the binding of the MBP–AT fusion protein and single AT domain
to substrates using MBP and bovine serum albumin as control proteins. Binding constants (Ka, per micromolar) were calculated and used to analyze the substrate specificity of the acyltransferase. We concluded that
there are many unrevealed new PKS gene clusters in marine sediments in the ECS. The acyltransferase is presumably an acetyltransferase
from a new PKS gene cluster. 相似文献
49.
Dr. Yongjun Zhou Prof. Patrícia Prediger Prof. Luiz Carlos Dias Dr. Annabel C. Murphy Prof. Peter F. Leadlay 《Angewandte Chemie (International ed. in English)》2015,54(17):5232-5235
Elaiophylin is an unusual C2‐symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non‐natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N‐acetylcysteamine thioester, produced a novel 16‐membered C2‐symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides. 相似文献
50.
Dr. Yuriy Rebets Dr. Elke Brötz Niko Manderscheid Dr. Bogdan Tokovenko Dr. Maksym Myronovskyi Prof. Dr. Peter Metz Dr. Lutz Petzke Dr. Andriy Luzhetskyy 《Angewandte Chemie (International ed. in English)》2015,54(7):2280-2284
Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin‐producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl‐carrier protein (ACP)‐encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3‐oxoadipyl‐CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3‐Oxoadipyl‐CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate. 相似文献