PHYTOMEDICINE
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2025
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IF 8.3
Qianwei Qu,Xingyu Huang,Zhenxin Zhu,Jun Wang,Mengmeng Zhao,Wenqiang Cui,Yadan Zheng,Yanyan Liu,Xueying Chen,Zhiyun Zhang,Na Dong,Chunliu Dong,Yanhua Li
Background Staphylococcus aureus is an opportunistic pathogen capable of readily forming biofilms, which can result in life-threatening infections involving different organs. Sanguinarine are benzo[c]phenanthridine alkaloids extracted from the Sanguinaria canadensis L. (Papaveraceae), which have a wide range of biological activities. Previous reports have shown that sanguinarine is able to induce an elevation of ROS to exert an anti- S. aureus effect. Nevertheless, the specific mechanism of action of sanguinarine against S. aureus biofilms remains unexplored. Purpose The objective of this study was to elucidate the target site of sanguinarine in S. aureus , as well as to investigate its mechanism of antimicrobial action and its interference with biofilm formation. Additionally, the study aimed to provide further evidence supporting the use of sanguinarine as an alternative to traditional antibiotics. Methods Initially, we assessed the in vitro anti- S. aureus properties of sanguinarine through a series of methodologies, including MIC assays, time-dependent assays, and resistance development studies. Secondly, we explored the antimicrobial mechanism of sanguinarine using TEM, membrane permeability assays, and membrane fluidity assays. Subsequently, the mechanism by which sanguinarine interferes with S. aureus biofilm formation was preliminarily analyzed in vitro . Additionally, the interaction between sanguinarine and imidazoleglycerol-phosphate dehydratase (IGPD) was investigated using bio-layer interferometry assays, circular dichroism spectroscopy, molecular docking, and site-directed mutagenesis to further elucidate the role of sanguinarine in biofilm disruption. Finally, the therapeutic efficacy of sanguinarine was evaluated in vivo using mouse models of biofilm and bacteremia. Results Herein, sanguinarine demonstrated notable antimicrobial properties and interfering effects on biofilm formation. Mechanistic investigations revealed that sanguinarine exerts its antimicrobial action by dissipating the proton motive force in bacteria and compromising the integrity and functionality of the cytoplasmic membrane. Furthermore, sanguinarine was found to regulate IGPD expression and inhibit L-histidine synthesis, thereby interfering S. aureus biofilm formation. Consequently, due to its polypharmacological effect, sanguinarine significantly reduced the S. aureus load in mouse organs and the formation of biofilm on the surface of implants in vivo without any resistance. Conclusions In this study, we demonstrated that sanguinarine can exert antibacterial and interfere with biofilm formation by disrupting the cell membrane of S. aureus and targeting IGPD. These findings suggest that sanguinarine holds potential for further development as a novel antibiotic to combat biofilm-associated infections.
研究领域:
微生物学
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药理学
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感染与免疫
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DOI
PubMed
高影响因子
ANALYTICAL BIOCHEMISTRY
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2016
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IF 2.243
Xiaoxia Shi,Shanshan Sha,Likun Liu,Xin Li,Yufang Ma
Mycobacterium tuberculosis dTDP- d -glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP- l -rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d - N -acetylglucosamine- l -rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions , and the kinetic parameters , were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.
研究领域:
微生物学
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药物开发
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生物化学
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2022
·Vol. 106
·期 9
·3625-3637
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IF 5.56
Wang Juan,Qu Ge,Xie Leipeng,Gao Chao,Jiang Yingying,Zhang Yi-Heng P. Job,Sun Zhoutong,You Chun
Abstract Dihydroxy-acid dehydratase (DHAD) plays an important role in the utilization of glycerol or glucose for the production of value-added chemicals in the in vitro synthetic enzymatic biosystem. The low activity of DHAD in the dehydration of glycerate to pyruvate hampers its applications in biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydration activity. A triple mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunken substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-fold higher reaction rates than the wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes. Key points • A screening method was established for the protein engineering of SsDHAD. • A R3 mutant of SsDHAD with 10-fold higher activity was obtained. • The R3 mutant exhibits higher productivity in the in vitro biosystems. Graphical abstract
研究领域:
酶学
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合成生物学
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生物催化
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DOI
PubMed
高影响因子
PLANT BIOTECHNOLOGY JOURNAL
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2025
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IF 10.5
Bo He,Yanhao Hu,Xu He,Wang Chen,Wei Yan,Xin Zang,Jiahai Zhou,Yonghao Ye
Dihydroxyacid dehydratase (DHAD), a key enzyme in the branched-chain amino acid (BCAA) synthesis pathway, is a promising herbicide target. Currently, no commercial herbicides target DHAD, and no inhibitors are in development. Structural analysis suggests that coordination with iron–sulfur clusters and hydrophobic interactions with Tyr215 and Phe181 are crucial for inhibitor binding. Based on this model, we designed and synthesised derivatives 4aa–4bb with a benzoxazinone ring. Compound 4bb effectively inhibited Arabidopsis seed germination at 50 μg/mL, reversible by BCAA supplementation. In vitro, 4bb showed a stronger binding affinity to DHAD ( K d = 83.18 μM) than AHAS ( K d = 132.70 μM). Compound 4bb exhibits minimal side effects on wheat seed germination at lower concentrations while ensuring high safety for rice at higher dosages during post-emergence treatment. Greenhouse tests revealed over 75% inhibition of broadleaf weeds such as Eclipta prostrata and Amaranthus retroflexus . The selectivity between crops and weeds is not due to species-specific DHAD differences. Safety assessments showed no significant toxicity to human cell lines and microorganisms. This study confirms DHAD as a viable herbicide target and offers a structural basis for optimising inhibitors, positioning 4bb as a lead compound for new herbicides.
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DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2021
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IF 3.493
Zhiji Chen,Feiying Mao,Haoteng Zheng,Qinjie Xiao,Zhihao Ding,Anming Wang,Xiaolin Pei
Aromatic nitriles are important structural motifs that frequently existed in pharmaceutical drugs. Due to the convenient synthesis of aldoximes from aldehydes, the dehydration of aldoximes to corresponding nitriles by aldoxime dehydratases (Oxds) is considered as a safe and robust enzymatic production route. Although the Oxd genes are widely distributed in microbial kingdom, so far less than ten Oxds were expressed and further characterized. In this study, we found 26 predicted putative Oxd genes from the GenBank database using a genome mining strategy. The Oxd gene from Pseudomonas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3). The amino acid sequence of OxdF1 shows high identities of 33∼85 % to other characterized Oxds, and contained a ferrous heme as the catalytic site. The optimum reaction pH and temperature of recombinant OxdF1 were 7.0 and 35 °C, respectively. OxdF1 was stable in pH 7.0 potassium phosphate buffer at 30 °C, and its half-life was approximately 3.8 h. OxdF1 can efficiently dehydrate aromatic and heterocyclic aldoximes to nitriles, such as 2-bromobenzaldoxime, 2-chloro-6-fluorobenzaldoxime, thiophene-2-carboxaldoxime, and pyridine-3-aldoxime. Therefore, the recombinant OxdF1 shows a potential application in the cyanide-free synthesis of aromatic nitriles.
研究领域:
生物化学
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酶学
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药物化学
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生物技术
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2022
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IF 5.895
Ya’nan Shi,Rongshan Li,Jie Zheng,Yubin Xue,Yong Tao,Bo Yu
Bio-based propionate attracts increasing attention owing to its green nature and specific food additive market. To date, the time-consuming and costly fermentation process by strict anaerobes makes propionate production not ideal. In this study, we designed a new route for propionate production, in which 1,2-propanediol was first dehydrated to propionaldehyde and then to propionate by taking advantage of the robust oxidization capacity of the Pseudomonas putida KT2440 strain. The high atom economy (0.97 g/g) in this proposed pathway is more advantageous than the previous l-threonine-derived route (0.62 g/g). The molecular mechanism of the extraordinary oxidation capacity of P. putida KT2440 was first deciphered. The propionate production was realized in P. putida KT2440 by screening suitable glycerol dehydratases and optimizing the expression to eliminate the formation of 1-propanol and the accumulation of the intermediate propionaldehyde. The engineered strain produced propionate with a molar conversion rate of >99% from 1,2-propanediol. A high titer of 46.5 g/L pure propionic acid with a productivity of 1.55 g/L/h and a mass yield of 0.96 g/g was achieved in fed-batch biotransformation. Thus, this study provides another idea for the production of high-purity bio-based propionate from renewable materials with high atom economy.
研究领域:
生物技术
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微生物学
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化学工程
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代谢工程
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DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2023
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IF 3.9
Shiming Tang,Yukun Chen,Daocheng Liao,Ying Lin,Shuangyan Han,Suiping Zheng
As an essential chemical, p- hydroxystyrene has attracted much attention and is widely used in the field of semiconductors. However, the current production methods have some disadvantages, such as low molar conversion yield, p- hydroxystyrene toxicity, and high substrate cost. To overcome these problems, an five enzymes cell-free cascade, consisting of alditol oxidase from Streptomyces coelicolor A3 (ScALDO), dehydratase from Paralcaligenes ureilyticus (PuDHT), tyrosine phenol lyase from Citrobacter freundii (CfTPL), tyrosine ammonia lyase from Rhodotorula glutinis (RgTAL) and phenylacrylate decarboxylase from Enterobacter sp. (EsPAD ) , was constructed for the de novo biosynthesis of p- hydroxystyrene from glycerol. Through process strategy optimization, a molar conversion yield of more than 84.2% was finally obtained from 10 mM crude glycerol. It is worth mentioning that the reaction system performed an excellent titer using 50 mM crude glycerol, and the p- hydroxystyrene concentration reached 30.32 mM, which exceeded the maximum titer of fermentation. This showed that our system is robust to p- hydroxystyrene and crude glycerol. In addition, a ‘two-step purification process’ was designed to produce pure p- hydroxystyrene. This is the first study focused on the cell-free biosynthesis of p- hydroxystyrene, and the highest conversion yield and titer thus far was obtained for p- hydroxystyrene de novo biosynthesis.
研究领域:
化学工程
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生物技术
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生物化学
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产品关联
DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2023
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IF 5
Pei Xiaolin,Xiao Qinjie,Feng Yumin,Chen Li,Yang Fengling,Wang Qiuyan,Li Nanxing,Wang Anming
Abstract Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. Graphical abstract
Key points • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.
研究领域:
生物化学
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生物催化
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有机化学
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香精香料行业
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酶学
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产品关联
DOI
PubMed
高影响因子
Journal of the American Chemical Society
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2024
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IF 14.4
Boyu Yang,Chao Li,Yongshuo Ren,Weichen Wang,Xiangxiang Zhang,Xiaojun Han
The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner–Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.
研究领域:
合成生物学
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代谢工程
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细胞生物学
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生物物理学
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生物化学
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产品关联
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PubMed
高影响因子
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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2020
·Vol. 117
·期 27
·15599-15608
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IF 9.412
Jiayi Liu,Yifeng Wei,Lianyun Lin,Lin Teng,Jinyu Yin,Qiang Lu,Jiawei Chen,Yuchun Zheng,Yaxin Li,Runyao Xu,Weixiang Zhai,Yangping Liu,Yanhong Liu,Peng Cao,Ee Lui Ang,Huimin Zhao,Zhiguang Yuchi,Yan Zhang
2(S)-dihydroxypropanesulfonate (DHPS) is a microbial degradation product of 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose), a component of plant sulfolipid with an estimated annual production of 1010 tons. DHPS is also at millimolar levels in highly abundant marine phytoplankton. Its degradation and sulfur recycling by microbes, thus, play important roles in the biogeochemical sulfur cycle. However, DHPS degradative pathways in the anaerobic biosphere are not well understood. Here, we report the discovery and characterization of two O2-sensitive glycyl radical enzymes that use distinct mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in sulfate- and sulfite-reducing bacteria catalyzes C–S cleavage to release sulfite for use as a terminal electron acceptor in respiration, producing H2S. DHPS-dehydratase (HpfG), in fermenting bacteria, catalyzes C–O cleavage to generate 3-sulfopropionaldehyde, subsequently reduced by the NADH-dependent sulfopropionaldehyde reductase (HpfD). Both enzymes are present in bacteria from diverse environments including human gut, suggesting the contribution of enzymatic radical chemistry to sulfur flux in various anaerobic niches.
研究领域:
微生物学
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环境科学
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生物地球化学
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生物化学
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DOI
PubMed
高影响因子
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