SENSORS AND ACTUATORS B-CHEMICAL
·
2016
·
IF 4.758
Xiaonan Wang,Ying Liu,Juan Zhang,Genxi Li
A new strategy to fabricate electrochemical biosensor is proposed based on the covalent capturing of molecules on electrode interface through oxime chemistry and the loading of signal probes via guanidinium-phosphate electronic interaction with a “covalent-like” stability. Taking aldolase as an example, enzyme analysis can be achieved through the covalent loading of the catalyzed products on the aminooxy modified gold electrode surface and subsequent immobilization of guanidinium functionalized sliver nanoparticles. Different from adolase substrate, the hydrolyzed products contain both carbonyl groups and phosphate acid groups in their structures. The carbonyl groups can chemoselectively react with aminoxy groups on the electrode surface to form oxime, resulting in the exposure of phosphate groups. Subsequently, signal probe can be loaded onto electrode surface through guanidinium recognition, giving a electrochemical current value. So the number of enzyme catalyzed products determines the amount of signal probes on the electrode surface, and aldolase activity can be analyzed. For the proposed method, the chemoselective oxime ligation and highly stable guanidinium recognition improve the accuracy, selectivity and stability of enzyme analysis. The established method may be extended to the analysis of other related substances containing carbonyl groups.
研究领域:
分析化学
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生物化学
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电化学
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生物传感器
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DOI
高影响因子
Journal of Biological Engineering
·
2020
·Vol. 14
·期 1
·1-10
·
IF 3.429
Xu Yingying,Meng Hao,Ren Jie,Zeng An-Ping
Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.
研究领域:
生物化学
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合成生物学
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代谢工程
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DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2025
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IF 7.3
Chaoqun Huang,Xuerui Jin,Yeyi Kan,Can Cui,Longchen Wei,Huayi Liu,Dawen Niu,Yunzi Luo
Nonnatural amino acids hold the promise of enhancing enzyme activity and bolstering the efficacy of drugs. Enzymatic synthesis emerges as a valuable adjunct to traditional synthetic methodologies, providing an alternative pathway for the generation of nonnatural amino acids. This study introduces a one-pot enzymatic strategy for the synthesis of aryl and heteroaryl γ-hydroxy-α-amino acids. The process involved the utilization of a newly identified aldolase from Streptomyces to catalyze the formation of C–C bond from the cost-effective fluorinated pyridine aldehyde substrate, and an α-transaminase sourced from Saccharomyces cerevisiae to transfer an amino group. After optimization, a 94% conversion efficiency was achieved, along with a product diastereoselectivity ratio of up to 13:87. Furthermore, the one-pot reaction system demonstrated efficacy across a broad spectrum of substrates, including fluorinated pyridines, quinolines, azabiphenyls, thiazoles, and phenyl derivatives. This study significantly broadened the scope of nonnatural amino acids containing heteroaromatic rings and diversified the array of enzymes accessible for the production of functionally diverse bioactive fluorinated compounds.
研究领域:
生物化学
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合成生物学
·
酶工程
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高影响因子
PHYTOMEDICINE
·
2024
·
IF 6.7
Xin-Rong Wang,Hua Zhong,Shan-Shan Ma,Ya-Hui Huang,Wei-Heng Xu,Yan Wang
Background The incidence of invasive fungal diseases (IFDs), represented by Candida albicans infection, is increasing year by year. However, clinically available antifungal drugs are very limited and encounter challenges such as limited efficacy, drug resistance, high toxicity, and exorbitant cost. Therefore, there is an urgent need for new antifungal drugs. Purpose This study aims to find new antifungal compounds from plants, preferably those with good activity and low toxicity, and reveal their antifungal targets. Methods In vitro antifungal activities of compounds were investigated using broth microdilution method, spot assay, hyphal growth assay and biofilm formation assay. Synergistic effects were assessed using broth microdilution checkerboard technique. In vivo antifungal activities were evaluated using Galleria mellonella and murine candidiasis models. Cytotoxicity of compounds was investigated using Cell Counting Kit-8 (CCK-8). Discovery and validation of antifungal targets of compounds were conducted by using monoallelic knockout library of C. albicans , haploinsufficiency profiling (HIP), thermal shift assay (TSA), enzyme inhibitory effect assay, molecular docking, and in vitro and in vivo antifungal studies. Results 814 plant products were screened, among which petroselinic acid (PeAc) was found as an antifungal molecule. As a rare fatty acid isolated from coriander ( Coriandrum sativum ), carrot ( Daucus carota ) and other plants of the Apiaceae family, PeAc had not previously been found to have antifungal effects. In this study, PeAc was revealed to inhibit the growth of various pathogenic fungi, exhibited synergistic effects with fluconazole (FLC), inhibited the formation of C. albicans hyphae and biofilms, and showed antifungal effects in vivo. PeAc was less toxic to mammalian cells. Fructose-1,6-bisphosphate aldolase (Fba1p) was identified as a target of PeAc by using HIP, TSA, enzyme inhibitory effect assay and molecular docking methods. PeAc exerted antifungal effects more effectively on fba1Δ/FBA1 than wild-type (WT) strain both in vitro and in vivo . Conclusions PeAc is an effective and low toxic antifungal compound. The target of PeAc is Fba1p. Fba1p is a promising target for antifungal drug development.
研究领域:
药理学
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微生物学
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真菌学
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植物化学
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药物发现
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DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2023
·
IF 4.4
Haidong Teng,Kaitong Chen,Lan Wang,Jianping Wu,Gang Xu
l -syn-3-[4-(methylsulfonyl)] phenylserine is a key industrial intermediate for the synthesis of florfenicol, thiamphenicol , and other products. l -Threonine aldolase can synthesize l -syn-3-[4-(methylsulfonyl)] phenylserine from glycine and 4-methylsulphonyl benzaldehyde directly. However, the selectivity of free l -threonine aldolase was difficult to meet the requirement of industrialization. Here, we selected the suitable site to insert His tag as immobilized site to improve the selectivity of l -threonine aldolase based on the molecular dynamics simulation and prior protein engineering. The phenanthroline was coupled to the surface of magnetic nanoparticles coated with silicon and thus enabled the selective and oriented immobilization of His-tagged l -threonine aldolase. Due to magnetic nanoparticles coated with silicon as supports, it was easy to separate the residual reactant and impurities from the immobilized enzyme by magnetic attraction. Moreover, the immobilization was combined with the purification of free enzymes through directional immobilization, which avoided heterozyme linking supports and improved the utilization efficiency of supports. Our study demonstrates an effective approach: how to analyze and select enzyme-targeted ligation sites in conjunction with previous modifications of engineered bacteria, and the immobilized enzyme obtained also provided a good candidate for the industrial production of l -syn-3-[4-(methylsulfonyl)] phenylserine.
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高影响因子
Molecular Catalysis
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2022
·
IF 5.089
Li-Chao Wang,Lian Xu,Bing-Mei Su,Xin-Qi Xu,Juan Lin
L- threo -4-methylsulfonylphenylserine ethyl ester [(2 S , 3 R )- 1d ], a key building block of florfenicol which synthesized by esterification using ethanol and L- threo -4-methylsulfonylphenylserine [(2 S , 3 R )- 1b ] as substrates. L-threonine aldolase (LTA) is a promising biocatalyst for producing (2 S , 3 R )- 1b through a one-step process taking 4-Methylsulphonyl benzaldehyde ( 1a ) and glycine as substrates under the mild condition. However, the moderate C β -stereoselective blocked the industrial application of LTA. To address this issue, rational design by combination with prereaction state molecular dynamics (MD) simulation and per-residue energy decomposition algorithm are employed to engineer LTA for enhancing C β -stereoselective. As a result, a triple mutant N16A/E98S/Y314R (Mu3) is screened out to produce (2 S , 3 R )- 1b with 93.7% de and 90.2% conversion under the 300 mM 1a substrate loading. Furthermore, esterification is applied to synthesize (2 S , 3 R )- 1d with over 99% chiral purity and 99% product purity. The success of this study provides new insights for the rational design of LTA with improved C β -stereoselectivity and proves a chemo-enzymatic route for green synthesis of (2 S , 3 R )- 1d .
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高影响因子
JOURNAL OF MEDICINAL CHEMISTRY
·
2022
·
IF 8.039
Wuqiang Wen,Hongxuan Cao,Yunyuan Huang,Jie Tu,Chen Wan,Jian Wan,Xinya Han,Han Chen,Jiaqi Liu,Li Rao,Chen Su,Chao Peng,Chunquan Sheng,Yanliang Ren
Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis, liquid chromatography–mass spectrometry, and the crystallographic structures of APO–CaFBA, CaFBA–G3P, and C157S–2a4 revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant C. albicans, and compound 2a11 can inhibit the growth of azole-resistant strains 103 with the MIC80 of 1 μg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.
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PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2016
·
IF 7.476
Xiaonan Wang,Meiwen Wang,Yuanyuan Zhang,Xiaocao Miao,Yuanyuan Huang,Juan Zhang,Lizhou Sun
A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate , thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe . The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p -phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability.
研究领域:
分析化学
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生物化学
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生物传感器
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电化学
·
纳米技术
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DOI
PubMed
高影响因子
Frontiers in Microbiology
·
2023
·
IF 5.2
Liu Quanzhen,Wei Guoguang,Yang Pengfan,Wang Chengyong,Chen Kequan,Ouyang Pingkai,Zhang Alei
N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources.
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PubMed
高影响因子
CHEMISTRY-A EUROPEAN JOURNAL
·
2021
·Vol. 27
·期 37
·9654-9660
·
IF 5.236
Lichao Wang,Lian Xu,Bingmei Su,Wei Lin,Xinqi Xu,Juan Lin
Graphical CASTing call : A semi-rational combinatorial active-site saturation test (CAST) was performed to improve the C β stereoselectivity of threonine aldolase. MD simulations suggested that the Y314R variant could interact with 4-methylsulfonyl benzaldehyde through hydrogen bonding to control the direction of entry of substrate, this increased the de value from 58 to 81 %. l -Threonine aldolase from Actinocorallia herbida ( Ah LTA) is an ideal catalyst for producing l - threo -4-methylsulfonylphenylserine [(2 S ,3 R )- 1 b ], a key chiral precursor for florfenicol and thiamphenicol. The moderate C β stereoselectivity is the main obstacle to the industrial application of Ah LTA. To address this issue, a combinatorial active-site saturation test (CAST) together with sequence conservatism analysis was applied to engineer the Ah LTA toward improved C β stereoselectivity. The optical mutant Y314R could asymmetrically synthesize l - threo -4-methylsulfonylphenylserine with 81 % diastereomeric excess ( de ), which is 23 % higher than wild-type Ah LTA. Molecular dynamic (MD) simulations revealed that the mechanism for the improvement in C β stereoselectivity of Y314R is due to the acylamino group of residues Arg314 controlling the orientation of substrate 4-methylsulfonyl benzaldehyde ( 1 a ) in the active pocket by directed interaction with the methylsulfonyl group; this leads to asymmetric synthesis of l - threo -4-methylsulfonylphenylserine. The success in this study demonstrates that direct control of substrates in an active pocket is an attract strategy to address the C β stereoselectivity problem of LTA and contribute to the industrial application of LTA.
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高影响因子
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