Molecular Catalysis
·
2019
·
IF 2.938
Guochao Xu,Wei Dai,Yue Wang,Lu Zhang,Zewen Sun,Jieyu Zhou,Ye Ni
Structure-guided rational design revealed the molecular switch manipulating the Prelog and anti-Prelog priorities of an NADPH-dependent alcohol dehydrogenase toward prochiral ketones with bulky and similar substituents. Synergistic effects of unconserved residues at 214 and 237 in small and large substrate binding pockets were proven to be vital in governing the stereoselectivity. The ee values of E214Y/S237A and E214C/S237 G toward (4-chlorophenyl)-(pyridin-2-yl)-methanone were 99.3% ( R ) and 78.8% ( S ) respectively. Substrate specificity analysis revealed that similar patterns were also found with (4’-chlorophenyl)-phenylmethanone, (4’-bromophenyl)-phenylmethanone and (4’-nitrophenyl)-phenylmethanone. This study provides valuable evidence for understanding the molecular mechanism on enantioselective recognition of prochiral ketones by alcohol dehydrogenase.
研究领域:
生物化学
·
酶学
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DOI
高影响因子
MOLECULAR BIOTECHNOLOGY
·
2021
·Vol. 63
·期 6
·491-501
·
IF 2.695
Tang Wanggang,Lian Chaoqun,Si Yu,Chang Jianrong
2,3-Butanediol dehydrogenase (BDH), also known as acetoin/diacetyl reductase, is a pivotal enzyme for the formation of 2,3-butanediol (2,3-BD), a chiral compound with potential roles in the virulence of certain pathogens. Here, a NAD(H)-dependent (2 R ,3 R )-BDH from Neisseria gonorrhoeae FA1090 (NgBDH), the causative agent of gonorrhoea, was functionally characterized. Sequence analysis indicated that it belongs to zinc-containing medium-chain dehydrogenase/reductase family. The recombinant NgBDH migrated as a single band with a size of around 45 kDa on SDS-PAGE and could be confirmed by Western blotting and mass spectrometry. For the oxidation of either (2 R ,3 R )-2,3-BD or meso -2,3-BD, the enzyme exhibited a broad pH optimum between pH 9.5 to 11.5. For the reduction of (3 R /3 S )-acetoin, the pH optimum was around 6.5. The enzyme could catalyze the stereospecific oxidation of (2 R ,3 R )-2,3-BD ( K m = 0.16 mM, k cat / K m = 673 s −1 · mM −1 ) and meso -BD ( K m = 0.72 mM, k cat / K m = 165 s −1 · mM −1 ). Moreover, it could also reduce (3 R /3 S )-acetoin with a K m of 0.14 mM and a k cat / K m of 885 s −1 · mM −1 . The results presented here contribute to understand the 2,3-BD metabolism in N. gonorrhoeae and pave the way for studying the influence of 2,3-BD metabolism on the virulence of this pathogen in the future.
研究领域:
微生物学
·
生物化学
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DOI
PubMed
高影响因子
Journal of Fungi
·
2023
·Vol. 9
·期 5
·512
·
IF 4.7
Xuan Wang,Xianhao Xu,Jiaheng Liu,Yanfeng Liu,Jianghua Li,Guocheng Du,Xueqin Lv,Long Liu
Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed aSaccharomyces cerevisiaestrain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed inS. cerevisiaeto realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol.Keywords:vitamin A;retinol;Saccharomyces cerevisiae;retinol dehydrogenase;olive oil
研究领域:
代谢工程
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生物技术
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微生物生物技术
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DOI
PubMed
高影响因子
LETTERS IN APPLIED MICROBIOLOGY
·
2015
·Vol. 61
·期 4
·390-396
·
IF 1.659
Chao H.,Wu B.,Shen P.
Increase in L‐serine production is of interest for industry. Here, we describe a metabolic engineering approach to increase the production of L‐serine in a methylotrophic bacterium. mxaF, the gene encoding the large subunit of a methanol dehydrogenase, was cloned from Methylobacterium sp. MB200 through transposon mutagenesis. Deletion of mxaF gene prevented the strain to grow on methanol, suggesting that mxaF is involved in methanol metabolism. Overexpression of mxaF gene in the strain MB200 resulted in a fivefold increase in methanol dehydrogenase activity compared to the wild‐type. Resting cell assays showed that the recombinant strain accumulated 6·6 mg ml−1 L‐serine in 72 h with 30 mg ml−1 wet cells from 50 mg ml−1 glycine and 50 mg ml−1 methanol, representing a 1·5‐fold increment for L‐serine production in contrast to the wild‐type strain. These results demonstrate that the potential for improving the production of L‐serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria.Significance and Impact of the StudyThe amount of L‐serine produced each year worldwide is relatively small compared with the amounts of the other amino acids and hence it is in great demand. Here, we describe a metabolic engineering approach to increase the production of L‐serine in a methylotrophic bacterium Methylobacterium sp. MB200. The result demonstrates that raising the output of L‐serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria.
研究领域:
微生物学
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生物技术
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代谢工程
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DOI
PubMed
高影响因子
Computational and Structural Biotechnology Journal
·
2021
·
IF 7.271
Jie Gu,Byu Ri Sim,Jiarui Li,Yangqing Yu,Lei Qin,Lunjie Wu,Yu Shen,Yao Nie,Yi-Lei Zhao,Yan Xu
Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24–7.81 fold increase in k cat /K m value compared with that of the wild type, when reacted with 8 aromatic ketones or β -ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network.
研究领域:
生物化学
·
蛋白质工程
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DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2021
·
IF 3.493
Yingying Zhou,Yu Wang,Xi Chen,Jinhui Feng,Min Wang,Qiaqing Wu,Dunming Zhu
Enzymatic stereospecific reduction of 17-oxosteroids offers an attractive approach to access 17β-hydroxysteroids of pharmaceutical importance. In this study, by adjusting the flexibility of α6-helix at the substrate entrance of the alcohol dehydrogenase from Ralstonia sp. (RasADH), the catalytic activity toward the stereospecific 17β-reduction of androstenedione was improved without sacrifice of the enantioselectivity. Among the mutants, F205I and F205A exhibited up to 623- and 523-fold improvement in catalytic efficiency , respectively, towards a range of different 17-oxosteroids compared to the wild-type enzyme. The corresponding 17β-hydroxysteroids were prepared in optically pure form with high space-time productivity and isolated yields using F205I as the biocatalyst, indicating that these mutants are promising biocatalysts for this useful transformation. These results suggest that modulating the flexibility of the active site lid offers an effective approach to engineer alcohol dehydrogenase for accommodating bulky steroidal substrates.
研究领域:
生物化学
·
药物合成
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DOI
高影响因子
Canadian Journal of Chemistry
·
2013
·Vol. 91
·期 11
·
IF 0.964
ChenMiao,LiuLiangliang,ChenXiaoqing
Alcohol dehydrogenase (ADH) is a key enzyme that converts ethanol into acetaldehyde. Its binders could be used to efficiently treat human toxic alcohol poisoning and suppress the acetaldehyde accumulation in alcohol hypersensitive alcoholics. In the present study, a new assay based on ADH-functionalized magnetic nanoparticles coupled with high-performance liquid chromatography − tandem mass spectrometry (HPLC−MS/MS) was developed for the rapid screening and identification of ADH binders. ADH was immobilized on silica-coated Fe3O4 magnetic nanoparticles via covalent bonds and the synthesized nanoparticles were characterized by TEM, XRD, FT-IR, and VSM. The amount of bound ADH onto magnetic nanoparticles was 80.6 μg/mg. The optimum temperature and pH for the enzymatic activity of immobilized ADH were 25 °C and 7.0, respectively. The relative activity of immobilized ADH remained 68.14% after 10 times of recycle, which exhibited good durability. Nine compounds with ADH-binding activity were screened and identified in Glycyrrhiza uralensis root extracts, among which seven compounds were first screened and identified. Experimental results proved that the proposed method could rapidly screen ADH binders from complex mixtures.
研究领域:
生物化学
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药物开发
·
分析化学
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DOI
高影响因子
Catalysis Science & Technology
·
2019
·Vol. 10
·期 1
·70-78
·
IF 5.726
Zeyu Yang,Hengwei Fu,Wenjie Ye,Youyu Xie,Qinghai Liu,Hualei Wang,Dongzhi Wei
Alcohol dehydrogenases (ADHs) together with the economical substrate-coupled cofactor regeneration system play a pivotal role in the asymmetric synthesis of chiral alcohols; however, severe challenges concerning the poor tolerance of enzymes to 2-propanol and the adverse effects of the by-product, acetone, limit its applications, causing this strategy to lapse. Herein, a novel ADH gene smadh2 was identified from Stenotrophomonas maltophilia by traditional genome mining technology. The gene was cloned into Escherichia coli cells and then expressed to yield SmADH2. SmADH2 has a broad substrate spectrum and exhibits excellent tolerance and superb activity to 2-propanol even at 10.5 M (80%, v/v) concentration. Moreover, a new thermostatic bubble column reactor (TBCR) system is successfully designed to alleviate the inhibition of the by-product acetone by gas flow and continuously supplement 2-propanol. The organic waste can be simultaneously recovered for the purpose of green synthesis. In the sustainable system, structurally diverse chiral alcohols are synthesised at a high substrate loading (>150 g L−1) without adding external coenzymes. Among these, about 780 g L−1 (6 M) ethyl acetoacetate is completely converted into ethyl (R)-3-hydroxybutyrate in only 2.5 h with 99.9% ee and 7488 g L−1 d−1 space–time yield. Molecular dynamics simulation results shed light on the high catalytic activity toward the substrate. Therefore, the high 2-propanol tolerance SmADH2 with the TBCR system proves to be a potent biocatalytic strategy for the synthesis of chiral alcohols on an industrial scale.
研究领域:
生物化学
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工业生物技术
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环境科学
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产品关联
DOI
高影响因子
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN
·
2018
·Vol. 92
·期 2
·275-282
·
IF 3.526
Sun Jiao,Zhang Dao,Zhao Wenli,Ji Qingmin,Ariga Katsuhiko
Optimized pore size is essential for maximizing the encapsulation efficiency of molecules in solid supports. In this work, we prepared porous silica nanosheets with mesopore in the size range from several to dozens of nanometers, which may provide suitable spaces for loading of various molecules. Without any surface modification, the porous silica nanosheets show excellent immobilization capability (27 wt%) of enzymes with large molecular sizes (>10 nm). An enhanced activity of alcohol dehydrogenase (ADH) in the porous silica nanosheets was also achieved in compared with free ADH or ADH in mesoporous silica nanoparticles with average pore size of 12 nm. In addition, this ADH-silica nanosheets system showed effective catalytic performance not only with pure ethanol, but also alcohol drinks with complicated gradients or after relative harsh treatments. The efficient supporting of this inorganic porous structure to enzymes might also lead to its potentials for other biocatalysis, biosensing and enzymatic determination of alcohols etc.
研究领域:
生物化学
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纳米材料科学
·
酶工程
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2012
·Vol. 97
·期 3
·1113-1120
·
IF 3.425
Jia Dongxu,Li Jianghua,Liu Long,Zhang Dongxu,Yang Yu,Du Guocheng,Chen Jian
A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris . The fragment was then amplified by polymerase chain reaction and inserted into the site between Eco RI and Not I sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae . The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using Sal I and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca 2+ and Mg 2+ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant ( K
m ) of 1.89 mg/mL and a maximum reaction rate ( V
max ) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris , and the achieved PVADH yield is the highest ever reported.
研究领域:
生物化学
·
分子生物学
·
微生物学
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产品关联
DOI
PubMed
高影响因子
TOXICOLOGY LETTERS
·
2023
·
IF 3.5
Huina Zhang,Shaowei Wang,Ya Chen,Jingjing Li,Yingna Zhai,Yunbing Tang,Huitao Li,Jianmin Sang,Hong Wang,Jieqiang Lv,Ren-shan Ge
Perfluoroalkylated carboxylic acids (PFCAs) are a subclass of man-made chemicals that have been widely used in industrial production and consumer products. As a result, PFCAs have been found to accumulate in the environment and bioaccumulate in organisms, leading to potential health and environmental impacts. This study investigated the inhibition of 11 PFCAs on gonadal 3β-hydroxysteroid dehydrogenases in humans, rats, and mice. We observed a V-shaped inhibition pattern against human granulosa (KGN) cell 3β-HSD2 starting from C9 (half-maximal inhibitory concentration, IC 50 , 100.8 μM) to C11 (8.92 μM), with a V-shaped turn. The same V-shaped inhibition pattern was also observed for PFCAs against rat testicular 3β-HSD1 from C9 (IC 50 , 50.43 μM) to C11 (6.60 μM). Mouse gonadal 3β-HSD6 was insensitive to the inhibition of PFCAs, with an IC 50 of 50.43 μM for C11. All of these PFCAs were mixed inhibitors of gonadal 3β-HSDs. Docking analysis showed that PFCAs bind to the nicotinamide adenine dinucleotide (NAD + )/steroid binding sites of these enzymes and bivariate correlation analysis showed that molecular length determines the inhibitory pattern of PFCAs on these enzymes. In conclusion, the carbon chain length determines the inhibitory strength of PFCAs on human, rat, and mouse gonadal 3β-HSDs, and the inhibitory strength of PFCAs against human and rat 3β-HSD enzymes shows V-shaped turn.
研究领域:
毒理学
·
内分泌学
·
环境健康科学
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 90
·期 9
·
IF 3.9
Yuan Li,Shu-Fang Li,Lin Zhang,Ya-Ping Xue,Yu-Guo Zheng
Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L−1 d−1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis.
研究领域:
生物技术
·
酶工程
·
生物化学
·
化学生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）