PROCESS BIOCHEMISTRY
·
2023
·
IF 4.4
Xinxin Wang,Xia Ke,Ximan Zhao,Qijie Ren,Jiahao Cui,Zhiqiang Liu,Yuguo Zheng
Understanding the mycobacteria degradation of sterol may facilitate strategies for the directed biotransformation of steroidal drug intermediates. Due to the long and complicated catabolic process, several key steps that involved in the C-C bond cleavage of steroidal side-chain remain unknown. In the present study, an aldolase SalA from Mycobacterium neoaurum was identified that was related to the synthesis of 3-oxo-4-pregnene-20-carboxyl aldehyde and 20-hydroxymethyl pregn-4-ene-3-one (4-HBC). As a member of the thiolase superfamily, the aldolase SalA-SalB is composed of a thiolase-like protein (SalA) and a DUF35 domain (SalB). As a result, the gene salA was determined that had special significance for the synthesis of 4-HBC in sterol metabolism by performing knockout and replenishment. Based on insights provided by the docking model and mutants analysis, the hypothesis of the catalytic mechanism was proposed. Furthermore, the deletion of salA absolutely eliminated the by-product 4-HBC and meanwhile the molar yield of ADD was increased by 10 %. The dominant role of SalA in the C24 steroidal side-chain cleavage as confirmed in this study provided a further clarification on the phytosterol degrading pathway in Mycobacterium neoaurum , which is of significant importance for the directed biotransformation between C19- and C22-steroids.
研究领域:
微生物学
·
生物化学
·
药物生物转化
✓ 已验证
产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2017
·
IF 6.32
Juan Zhang,Xiaonan Wang,Tingjun Chen,Chang Feng,Genxi Li
In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of a lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility and it can be applied for real sample analysis. What is more, because the lipid bilayer is the universal basis for cell-membrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.
研究领域:
生物化学
·
分析化学
✓ 已验证
DOI
PubMed
高影响因子
Journal of Pharmaceutical Analysis
·
2024
·
IF 6.1
Xin Yang,Guang-Yuan Ma,Xiao-Qiang Li,Na Tang,Yang Sun,Xiao-Wei Hao,Ke-Han Wu,Yu-Bo Wang,Wen Tian,Xin Fan,Zezhi Li,Caixia Feng,Xu Chao,Yu-Fan Wang,Yao Liu,Di Li,Wei Cao
Hepatocellular carcinoma (HCC) expresses abundant glycolytic enzymes and displays comprehensive glucose metabolism reprogramming. Aldolase A (ALDOA) plays a prominent role in glycolysis; however, little is known about its role in HCC development. In the present study, we aim to explore how ALDOA is involved in HCC proliferation. HCC proliferation was markedly suppressed both in vitro and in vivo following ALDOA knockout, which is consistent with ALDOA overexpression encouraging HCC proliferation. Mechanistically, ALDOA knockout partially limits the glycolytic flux in HCC cells. Meanwhile, ALDOA translocated to nuclei and directly interacted with c-Jun to facilitate its Thr93 phosphorylation by P21-activated protein kinase; ALDOA knockout markedly diminished c-Jun Thr93 phosphorylation and then dampened c-Jun transcription function. A crucial site Y364 mutation in ALDOA disrupted its interaction with c-Jun, and Y364S ALDOA expression failed to rescue cell proliferation in ALDOA deletion cells. In HCC patients, the expression level of ALDOA was correlated with the phosphorylation level of c-Jun (Thr93) and poor prognosis. Remarkably, hepatic ALDOA was significantly upregulated in the promotion and progression stages of diethylnitrosamine-induced HCC models, and the knockdown of ALDOA strikingly decreased HCC development in vivo . Our study demonstrated that ALDOA is a vital driver for HCC development by activating c-Jun-mediated oncogene transcription, opening additional avenues for anti-cancer therapies.
研究领域:
肿瘤学
·
分子生物学
·
癌症研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 90
·期 8
·
IF 3.9
Yating Li,Tong Xu,Yanqin Tu,Tong Li,Yifeng Wei,Yan Zhou
Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn2+-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP+-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO2 and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.
研究领域:
微生物学
·
生物化学
·
代谢途径
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2020
·
IF 3.448
Wenlong Zheng,Kaitong Chen,Sai Fang,Xiuli Cheng,Gang Xu,Lirong Yang,Jianping Wu
A number of organic synthesis involve threonine aldolase (TA), a pyridoxal phosphate (PLP)-dependent enzyme. Although the addition of exogenous PLP is necessary for the reactions, it increases the cost and complicates the purification of the product. This work constructed a PLP self-sufficient biocatalysis system for TA, which included an improvement of the intracellular PLP level and co-immobilization of TA with PLP. Engineered strain BL-ST was constructed by introducing PLP synthase PdxS/T to Escherichia coli BL21(ED3). The intracellular PLP concentration of the strain increased approximately fivefold to 48.5 μmol/g DCW . l -TA, from Bacillus nealsonii ( Bn LTA), was co-expressed in the strain BL-ST with PdxS/T, resulting in the engineered strain BL- Bn LTA-ST. Compared with the control strain BL- Bn LTA (254.1 U/L), the enzyme activity of the strain BL- Bn LTA-ST reached 1518.4 U/L without the addition of exogenous PLP. An efficient co-immobilization system was then designed. The epoxy resin LX-1000HFA wrapped by polyethyleneimine (PEI) acted as a carrier to immobilize the crude enzyme solution of the strain BL- Bn LTA-ST mixed with an extra 100 μM of exogenous PLP, resulting in the catalyst HFA PEI - Bn LTA-STPLP 100. HFA PEI - Bn LTA-STPLP 100 exhibited a half-life of approximately 450 h, and the application of the catalyst in the continuous biosynthesis of 3-[4-(methylsulfonyl) phenyl] serine had more than 180 batch reactions (>60% conv ) without the extra addition of exogenous PLP. The excellent compatibility and stability of the system were further confirmed by other TAs. This work introduced a PLP self-sufficient biocatalysis system that can reduce the cost of PLP and contribute to the industrial application of TA. In addition, the system may also be applied in other PLP-dependent enzymes.
研究领域:
生物化学
·
酶工程
·
工业微生物学
✓ 已验证
DOI
PubMed
高影响因子
Catalysis Science & Technology
·
2026
·
IF 4
Can Cui,Xuerui Jin,Yunzi Luo
The construction of valuable fluorinated molecules relies critically on the extension of fluorine-containing modules, fostering innovations across medicinal, material, and agricultural fields. Aldolases serve as efficient biocatalysts for stereoselective carbon-carbon bond formation between an aldehyde and a defined electrophilic partner, enabling controlled carbon-chain elongation. However, the efficiency of this aldol addition is often limited by the poor aqueous solubility of the aldehyde substrate. The presence of additional organic solvents can raise the dissolved substrate concentration and accelerate mass transfer, thereby enhancing overall conversion, but may lead to the loss of enzyme activity. To address this issue, we wrapped aldolase in a thin polymer shell by a two-step, in-situ encapsulation. The nanocapsule shields the protein from DMSO or acetonitrile while letting substrates and products pass freely. This modification strategy significantly improved the catalytic efficiency towards nine fluorobenzaldehyde derivatives, with an increase ranging from 1.45-to 21.25-fold. These findings provided a promising strategy for advancing biocatalysis in organic solvent-rich environments.
✓ 已验证
DOI
高影响因子
CHEMICAL ENGINEERING SCIENCE
·
2020
·
IF 3.871
Li-Chao Wang,Lian Xu,Xin-Qi Xu,Bing-Mei Su,Juan Lin
L-threonine aldolase (LTA) is a PLP-dependent enzyme that can reversibly catalyze aldol reaction of glycine and acetaldehyde to produce β -hydroxy- α -amino acids. In the present work, a putative lta gene from Actinocorallia herbida (AhLTA) was mined and over-expressed in Escherichia coli BL21 (DE3). The substrate spectrum assay indicated that AhLTA only used glycine as donor substrate and tolerated a wild range of aromatic aldehydes as acceptor substrates. It was found that the type and position of substituents in the aromatic aldehydes exerted a significant impact on the activity and stereoselectivity at β -carbon of AhLTA. Among those substrates, AhLTA could catalyze glycine and 4-methylsulphonyl benzaldehyde ( 14a ) to produce L- threo -4-methylsulfonylphenylserine ((2 S ,3 R )- 14b ) with high conversion (94.4%) and moderate stereoselectivity (19% de ). By conditional optimization, the de value of (2 S , 3 R )- 14b was improved to 61% and the conversion was 75%. Taken together, our study suggested that AhLTA might be a promising catalyst for producing chiral β -hydroxy- α -amino acids.
研究领域:
生物化学
·
酶学
·
有机化学
·
生物催化
✓ 已验证
产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Qianqian Jiang,Qi Chu,Qi Zhang,Chaoqiang Wu,Tongqing Tang,Xin Wang,Feifei Chen,Alei Zhang,Kequan Chen
Adol condensation mediated by L-threonine aldolase (LTA) is a promising route to synthesize β-hydroxy-α-amino acids, such as the anti-Parkinson's disease drug L- threo -3,4-dihydroxyphenylserine (L- threo -DOPS; droxidopa). However, the biocatalytic approach generally suffered from poor stereoselectivity at the C β chiral center. Herein, several LTAs from diverse microorganisms were examined for the stereoselective synthesis of L- threo -DOPS, and an LTA from Firmicutes bacterium ( Fb LTA) was identified with relatively high stereoselectivity. A semi-rational design was then employed to further enhance the stereoselectivity, resulting in the identification of beneficial mutations and the generation of beneficial mutants with markedly elevated stereoselectivity (up to 80 % de) over the wild-type (23 % de). Molecular dynamics (MD) simulation revealed that the significant improvement of stereoselectivity could be attributed to the structural reconstitution of substrate tunnels. Finally, the best mutant was applied to a scaled-up reaction, affording 1.3 g/L yield of L- threo -DOPS with 80 % de. This study provides new insights into the stereoselectivity control of LTAs and potential engineered LTAs for the manufacture of L- threo -DOPS.
研究领域:
生物化学
·
合成生物学
·
药物开发
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Sai Fang,Zhe Wang,Lanxin Xiao,Yan Meng,Yixuan Lei,Tianxin Liang,Yuhuan Chen,Xiaoshu Zhou,Gang Xu,Lirong Yang,Wenlong Zheng,Jianping Wu
l -threonine aldolase (LTA) catalyzes the synthesis of β -hydroxy- α -amino acids, which are important chiral intermediates widely used in the fields of pharmaceuticals and pesticides. However, the limited thermostability of LTA hinders its industrial application. Furthermore, the trade-off between thermostability and activity presents a challenge in the thermostability engineering of this enzyme. This study proposes a strategy to regulate the rigidity of LTA's V-shaped subunit by modifying its opening and hinge regions, distant from the active center, aiming to mitigate the trade-off. With LTA from Bacillus nealsonii as targeted enzyme, a total of 25 residues in these two regions were investigated by directed evolution. Finally, mutant G85A/M207L/A12C was obtained, showing significantly enhanced thermostability with a 20 °C increase in T 50 60 to 66 °C, and specific activity elevated by 34 % at the optimum temperature. Molecular dynamics simulations showed that the newly formed hydrophobicity and hydrogen bonds improved the thermostability and boosted proton transfer efficiency. This work enhances the thermostability of LTA while preventing the loss of activity. It opens new avenues for the thermostability engineering of other industrially relevant enzymes with active center located at the interface of subunits or domains.
研究领域:
生物化学
·
酶工程
·
蛋白质工程
✓ 已验证
DOI
PubMed
高影响因子
ADVANCED SYNTHESIS & CATALYSIS
·
2022
·Vol. 364
·期 24
·4363-4370
·
IF 5.981
Sai Fang,Haoran Yu,Lanxin Xiao,Zhe Wang,Yixuan Lei,Gang Xu,Lirong Yang,Wenlong Zheng,Jianping Wu
l -threonine aldolase ( L TA) is a vital tool for the production of β -hydroxy- α -amino acids, important pharmaceutical intermediates with two chiral centres. However, the trade-off between activity and diastereoselectivity seriously hinders the application of LTA. Here, microenvironment of the proton transfer was regulated to improve the enzyme activity while avoiding the loss of diastereoselectivity. A combinatorial active-site saturation test (CAST) strategy was applied to engineer the microenvironment of the key histidines H86 and H128 involved in proton transfer. Except for the amino acid residues tunning diastereoselectivity, a total of 18 (9+9) residues lining around H86 and H128 were investigated. As a result, two variants, RS1-T92V and RS1-E123R, were obtained with specific activity from 9.61 U/mg to 11.24 U/mg and 14.41 U/mg, respectively. By combinatorial mutagenesis, a double-point mutant RS1-VR (T92V/E123R) was obtained with specific activity reaching 18.65 U/mg that was two-fold of the original strain (RS1). Notably, the mutant RS1-VR remained a high de value of 94.21%. Molecular dynamics (MD) simulations provided insights into the mechanism of activity-diastereoselectivity trade-off. The improvement of microenvironment contributes to reduce the swing amplitude of the side chain of H86, resulting in the proton transfer more efficient. This work provides a strategy of regulating the proton transfer microenvironment for counteracting the trade-off between activity and diastereoselectivity in protein engineering.
研究领域:
生物催化
·
酶工程
·
计算化学
·
蛋白质工程
✓ 已验证
产品关联
DOI
高影响因子
BIOORGANIC & MEDICINAL CHEMISTRY
·
2022
·
IF 3.461
Baoqin Cai,Marco Bocola,Ameng Zhou,Fenshuai Sun,Qing Xu,Jiadong Yang,Tianran Shen,Zhaoqi Zhang,Lei Sun,Yaoyao Ji,Yong Koy Bong,Thomas Daussmann,Haibin Chen
l -Threonine aldolases (LTAs) employing pyridoxal phosphate (PLP) as cofactor can convert low-cost achiral substrates glycine and aldehyde directly into valuable β-hydroxy-α-amino acids such as (2 R ,3 S )-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid (( R,S )-AHNPA), which is utilized broadly as crucial chiral intermediates for bioactive compounds. However, LTAs’ stereospecificity towards the β carbon is rather moderate and their activity and stability at high substrate load is low, which limits their industrial application. Here, computer-aided directed evolution was applied to improve overall activity, selectivity and stability under desired process conditions of a l -threonine aldolase in the asymmetric synthesis of ( R,S )-AHNPA. Selectivity and stability determining regions were computationally identified for structure-guided directed evolution of LTA-variants under efficient biocatalytic process conditions using 40% ethanol as cosolvent. We applied molecular modeling to rationalize selectivity improvement and design focused libraries targeting the substrate binding pocket, and we also used MD simulations in nonaqueous process environment as an effective and promising method to predict potential unstable loop regions near the tetramer interface which are hot-spots for cosolvent resistance. An excellent LTA variant EM-ALDO031 with 18 mutations was obtained, which showed ∼ 30-fold stability improvement in 40% ethanol and diastereoselectivity ( de ) raised from 31.5% to 85% through a three-phase evolution campaign. Our fast and efficient data-driven methodology utilizing a combination of experimental and computational tools enabled us to evolve an aldolase variant to achieve the target of 90% conversion at up to 150 g/L substrate load in 40% ethanol, enabling the biocatalytic production of β-hydroxy-α-amino acids from cheap achiral precursors at multi-ton scale.
研究领域:
生物化学
·
酶工程
·
计算生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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
·
生物化学
·
电化学
·
生物传感器
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）