1.Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot.
Piyu KE ; Jun HUANG ; Sheng HU ; Weirui ZHAO ; Changjiang LÜ ; Kai YU ; Yinlin LEI ; Jinbo WANG ; Lehe MEI
Chinese Journal of Biotechnology 2016;32(1):31-40
Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.
Glutamate Decarboxylase
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metabolism
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Half-Life
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Industrial Microbiology
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Lactobacillus brevis
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enzymology
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Mutagenesis, Site-Directed
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Mutation
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Temperature
2.Correlation analysis of GSTO1 expression in cervical cancer tissue with prognosis and the impact of N-glycosylation on the malignant biological behavior of cervical cancer
Panpan YU ; Ping YANG ; Qianyu SUN ; Weirui GAO ; Zouyu ZHAO ; Chongfeng SUN
Acta Universitatis Medicinalis Anhui 2023;58(12):2002-2010
Objective To investigate the expression of(glutathione S-Transferase Omega-1,GSTO1)in cervical cancer tissue and its correlation with patient survival time,and to explore the impact of GSTO1 N-glycosylation on proliferation,migration,invasion,and epithelial-mesenchymal transition of cervical cancer.Methods By using immunohistochemistry,the expression levels of GSTO1 in tumor cells of 82 cervical cancer patients were detected,and the correlation between GSTO1 expression and clinical pathological features was analyzed.Kaplan-Meier meth-od was used to plot survival curves and evaluate the impact of GSTO1 expression in cervical cancer tissues on pa-tient survival time.Univariate and multivariate Cox regression analyses were performed to assess the independent prognostic factors influencing cervical cancer prognosis.The NetNGlyc 1.0 Server database predicted potential N-glycosylation modification sites of GSTO1(Asn55,Asnl35,Asn190).The cervical cancer cells(HeLa)were transfected with GSTO1 N-glycosylation site mutation vectors at positions 55,135,and 190,as well as GSTO1 wild-type vector and empty vector.Stable transfected cells were selected using puromycin.Western blot experi-ments were performed to assess the effectiveness of lentiviral interference.The effects of GSTO1 N-glycosylation site mutations on proliferation,migration,and invasion of HeLa cells were evaluated using EdU proliferation assay,wound healing assay,and Transwell assay.The effect of GSTO1 N-glycosylation site mutations on the epithelial-mesenchymal transition of HeLa cells was detected using the Western blot experiment.Results Immunohistochem-istry results revealed high expression of GSTO1 in cervical cancer tissues.The expression rate of GSTO1 was signifi-cantly higher in cervical cancer tissues with deep stromal invision≥1/2,lymphovascular space invasion,and lymph node metastasis(P<0.05).Moreover,high expression of GSTO1 was associated with poorer overall surviv-al.After N-glycosylation site-specific mutation of GSTO1,the cell count of proliferation,migration,and invasion in HeLa cells significantly decreased(P<0.05).The Western blot results showed that N-glycosylation site mutation of GSTO1 significantly inhibited the epithelial-mesenchymal transition of HeLa cells.Conclusion The expression of GSTO1 in cervical cancer tissues is associated with stromal infiltration depth,lymphovascular space invasion and lymph node metastasis,and it is also correlated with shorter patient survival time.Site-specific mutations in GSTO1 N-glycosylation significantly inhibit the proliferation,migration and epitheli al-mesenchymal transition of HeLa cells.
3.Deletion of a dynamic surface loop improves thermostability of (R)-selective amine transaminase from Aspergillus terreus.
Dongfang XIE ; Changjiang LV ; Hui FANG ; Weikang YANG ; Sheng HU ; Weirui ZHAO ; Jun HUANG ; Lehe MEI
Chinese Journal of Biotechnology 2017;33(12):1923-1933
Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.
4.Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline.
Hui FANG ; Changjiang LÜ ; Yujiao HUA ; Sheng HU ; Weirui ZHAO ; Wenji FANG ; Kui SONG ; Jun HUANG ; Lehe MEI
Chinese Journal of Biotechnology 2019;35(4):636-646
Glutamate decarboxylase, a unique pyridoxal 5'-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T₅₀ ¹⁵) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.
Glutamate Decarboxylase
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Glutamic Acid
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Lactobacillus brevis
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Molecular Dynamics Simulation
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Proline
5.Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability.
Tingting CAI ; Jiaren CAO ; Shuai QIU ; Changjiang LYU ; Fangfang FAN ; Sheng HU ; Weirui ZHAO ; Lehe MEI ; Jun HUANG
Chinese Journal of Biotechnology 2023;39(6):2126-2140
ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Transaminases/chemistry*
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Molecular Docking Simulation
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Amines/chemistry*
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Pyruvic Acid/metabolism*
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Enzyme Stability