Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum
Abstract
:1. Introduction
2. Results
2.1. Differential Expression Pattern of Four SlCLH Genes in Various Tissues
2.2. Various cis-Acting Elements Related to Abiotic Stresses and Phytohormone Response Are Present in the Promoters of Four SlCLH Genes
2.3. Analysis of Amino Acid Sequences of SlCLHs
2.4. Expression and Purification of Recombinant SlCLHs in E. coli
2.5. Optimal Temperature and pH for the Activity of the Recombinant SlCLHs
2.6. Enzyme Kinetics of Recombinant SlCLHs
2.7. Identification of Putative Catalytic Triad
3. Discussion
3.1. Different Expression Pattern of SlCLHs Suggest That They May Have Distinct Roles in Various Tissues
3.2. SlCLHs Belong to the α/β Hydrolase Superfamily, but Show Different Physiological and Biochemical Characteristics
3.3. The Role of the Predicted N-Terminal Processing Sequences in the Activity of Recombinant SlCLHs
4. Materials and Methods
4.1. Plant Materials
4.2. RNA Isolation
4.3. Gene Expression Analysis
4.4. Protein Expression and Purification
4.5. Identification of the Putative Catalytic Triad
4.6. Sequence Analysis
4.7. In Silico Analysis of Cis-Elements in Promoters of SlCLH Genes
4.8. Chl Dephytylation Activity Assay
4.9. Analysis of Biochemical Characteristics of Recombinant SlCLHs
4.10. Enzyme Kinetic Assay
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Source | Accession No. | Molecular Weight | pI | Estimated Half-Life (Hours) | Instability Index | Aliphatic Index | GRAVY | ||
---|---|---|---|---|---|---|---|---|---|
Mammalian Reticulocytes, In Vitro | Yeast, In Vivo | E. coli, In Vivo | |||||||
SlCLH1 | Solyc06g053980.2.1 | 34,048.30 | 6.86 | 30 h | >20 h | >10 h | 39.87 | 93.08 | −0.019 |
SlCLH2 | Solyc09g065620.2.1 | 33,706.42 | 9.02 | 30 h | >20 h | >10 h | 44.59 | 98.76 | 0.081 |
SlCLH3 | Solyc09g082600.1.1 | 34,600.81 | 8.17 | 30 h | >20 h | >10 h | 40.00 | 90.98 | −0.028 |
SlCLH4 | Solyc12g005300.1.1 | 37,257.12 | 9.14 | 30 h | >20 h | >10 h | 41.32 | 86.03 | −0.146 |
Recombinant SlCLHs | Substrate | Vmax | Km (μM) | kcat (10−3 s−1) | kcat/Km |
---|---|---|---|---|---|
(10−3 μmoL mg−1 min−1) | (10−5 s−1μM−1) | ||||
SlCLH1 | Chl a | 18.93 | 30.42 | 23.36 | 76.79 |
Chl b | 23.86 | 48.52 | 29.45 | 60.68 | |
Phein a | 2.61 | 302.50 | 3.22 | 1.06 | |
SlCLH2 | Chl a | 1.96 | 140.17 | 2.41 | 1.72 |
Chl b | 2.22 | 109.03 | 2.73 | 2.50 | |
SlCLH3 | Chl a | 0.41 | 258.86 | 0.51 | 0.20 |
Chl b | 0.59 | 295.32 | 0.73 | 0.25 |
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Liu, G.; Meng, X.; Ren, Y.; Zhang, M.; Chen, Z.; Zhang, Z.; Pang, X.; Zhang, X. Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum. Int. J. Mol. Sci. 2022, 23, 11716. https://doi.org/10.3390/ijms231911716
Liu G, Meng X, Ren Y, Zhang M, Chen Z, Zhang Z, Pang X, Zhang X. Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum. International Journal of Molecular Sciences. 2022; 23(19):11716. https://doi.org/10.3390/ijms231911716
Chicago/Turabian StyleLiu, Guangyuan, Xue Meng, Yujun Ren, Min Zhang, Ziqing Chen, Zhaoqi Zhang, Xuequn Pang, and Xuelian Zhang. 2022. "Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum" International Journal of Molecular Sciences 23, no. 19: 11716. https://doi.org/10.3390/ijms231911716