Part:BBa_K5288000
Pyrroline-5-carboxylate synthetase
Description
The bio brick Delta-Pyrroline-5-carboxylate synthetase consists of two domains protein fused with a 6x His-tag to facilitate the purification process; N-terminal Glutamate kinase domain which catalyzes glutamate phosphorylation, and a C-terminal γ-glutamyl phosphate reductase [1], [2], [3], [4], [5], [6]. The enzyme has a length of 667 aa and a molecular weight of 72.36 kDa. This part was adapted to the Golden Gate cloning method. Also, the 6-HisTag is located at the C-terminal site, to facilitate the purification process.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 137
Illegal AgeI site found at 124 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Hydric and heat stress occur when plants are exposed to drought conditions or extreme heat. This stress can reduce crop yields to nearly half of their typical production levels, often resulting in poor or unusable crops. Consequently, this creates a scarcity of essential nutrients in staple foods. Current treatments involve direct soil applications like compost, humic acids, and hydrogels, but these methods can be harmful if misapplied or are not environmentally sustainable [7], [8], [9]. Our team has been researching safer, more effective, and easy-to-implement alternatives to enhance crop performance under these challenging conditions.
The principle behind the biostimulant relies on the biosynthesis of osmolytes and osmoprotectants . Similar to electrolytes, osmolytes contribute to homeostasis maintenance. They provide the driving gradient for water uptake and maintain cell turgor by osmotic adjustment and redox metabolism to remove excess reactive oxygen species (ROS) and reestablish the cellular redox balance. Also, protecting cellular machinery from osmotic stress and oxidative damage [10]. Related to osmolytes, osmoprotectants can protect cells against osmotic stress and high salinity conditions. These compounds protect cells by stabilizing proteins, maintaining membrane integrity, and scavenging reactive oxygen species [11].
Proline biosynthesis happens naturally in plant cells by a multi-enzymatic process. The main pathway in plants under stress conditions is the glutamate pathway [12], [13], [14]. Glutamate is phosphorylated by γ-glutamyl kinase yielding γ-glutamyl phosphate, which then is converted by the enzyme γ-glutamyl phosphate reductase to glutamate γ-semialdehyde. Commonly in plants, this reaction is catalyzed by a single bifunctional enzyme, P5C synthase (P5CS) [2], [4]. Glutamate γ-semialdehyde transforms to δ1-pyrroline-5-carboxylate (P5C) after a spontaneous cyclization. Finally, P5C is reduced by the cofactor NAD(P)H to yield L-proline and the oxidized cofactor NAD(P)+. This last step is catalyzed by the enzyme P5C reductase (P5CR) [5], [12], [15], [16]. P5CS is the rate-limiting enzyme and determines the biosynthesis of proline [17]. Biosynthetic enzymes (P5CS and P5CR) are found in the cytosol; therefore, this is where proline biosynthesis takes place. [3], [14], [18].
Pyrroline-5-carboxylate synthetase (P5CS) is common among life, as it serves a big role in proline biosynthesis to combat abiotic stress, especially in plants [12], [13], [14]. It is composed of two domains, γ-glutamyl kinase and γ-glutamyl phosphate reductase [3], [4], [5], [6]. In bacteria and most prokaryotic organisms, these two domains are produced as single proteins to synthesize Pyrroline-5-carboxylate. However, in higher eukaryotes; mammals, plants, and some unicellular eukaryotes; both enzymatic activities are fused in a single enzyme P5CS [1], [4], [19]. Furthermore, P5CS is commonly present on two isogenes: P5CS-1 takes the main role in proline biosynthesis and accumulation under abiotic stress. [1], [3], [5], [18], [20]. The importance of the duplication of this isogene has been studied within its evolutionary outcome, as a recent study made in 2024 revealed that approximately 75% of plant species have the duplicated gene of P5CS [1].
Plant P5CS-1's are commonly composed of two domains. N-terminal γ-glutamyl kinase and C-terminal γ-glutamyl phosphate reductase [3], [4], [5], [6]. Specifically, this biobrick is the coding sequence for P5CS-1 from Zea mays (LOC100280719).
The N-terminal domain of P5CS is composed of the enzyme γ-glutamyl kinase (GK) [1], [3], [4], [5], [6]. GK catalyzes the phosphorylation of glutamate to synthesize the highly unstable product: glutamyl-5-phosfate [21]. Commonly on prokaryotes GK, which is encoded by the ProB gene, is composed of an N-terminal kinase domain and a C-terminal PUA domain that is found on RNA-modifying enzymes. The PUA domain is not necessary for the functioning of the enzyme [1], [21], [22]. In plants and mammals, the GK domain does not include the PUA domain [1], [22].
The C-terminal domain of P5CS is composed of the γ-glutamyl phosphate reductase (GPR) [1], [3], [4], [5], [6]. GPR catalyzes the reduction of glutamyl-5-phosphate into glutamate γ-semialdehyde [23]. GPR consists of three domains, the first two act as the binding factor domain and the catalytic domain, both present a basic α/β architecture. The third domain acts as an oligomerization domain, which links the other two domains [24].
We intend to use the P5CS-1 as a key enzyme for the biosynthesis of the osmolyte and osmoprotectant; proline. As mentioned previously, P5CS-1 catalyzes the first step in the biosynthesis of proline. With this, we intend to ensure a better resistance to drought and caloric stress on corn crops. Proline aids cell turgor maintenance and osmotic balance in plants under this stress. Furthermore, proline stabilizes cell membranes, prevents electrolyte leakage, and protects vegetal cells from oxidative damage [25]. This will lead to a novel non-transgenic or synthetic solution to losses on corn production globally due to abiotic stress. It has been demonstrated that, in high doses, exogenous supplementation of proline in crops leads to diminished growth and development [26].
References
[1] J. P. C. Filgueiras, M. Zámocký, and A. C. Turchetto-Zolet, “Unraveling the evolutionary origin of the P5CS gene: a story of gene fusion and horizontal transfer,” Front. Mol. Biosci., vol. 11, p. 1341684, Apr. 2024, doi: 10.3389/fmolb.2024.1341684.
[2] Y. Kawji, “An Investigation of the Proline Biosynthesis Pathway in Response to Osmotic Stress in Tigriopus californicus,” Honors Theses, Apr. 2020, Available: https://repository.lsu.edu/honors_etd/822
[3] G. Sabbioni, D. Funck, and G. Forlani, “Enzymology and Regulation of δ1-Pyrroline-5-Carboxylate Synthetase 2 From Rice,” Front. Plant Sci., vol. 12, p. 672702, Sep. 2021, doi: 10.3389/fpls.2021.672702.
[4] A. N. Bogner, K. M. Stiers, and J. J. Tanner, “Structure, biochemistry, and gene expression patterns of the proline biosynthetic enzyme pyrroline-5-carboxylate reductase (PYCR), an emerging cancer therapy target,” Amino Acids, vol. 53, no. 12, pp. 1817–1834, Dec. 2021, doi: 10.1007/s00726-021-02999-5.
[5] L. Zhang and D. F. Becker, “Connecting proline metabolism and signaling pathways in plant senescence,” Front. Plant Sci., vol. 6, Jul. 2015, doi: 10.3389/fpls.2015.00552.
[6] P. Singh, A. Tiwari, S. P. Singh, and R. K. Asthana, “Proline biosynthesizing enzymes (glutamate 5-kinase and pyrroline-5-carboxylate reductase) from a model cyanobacterium for desiccation tolerance,” Physiol. Mol. Biol. Plants, vol. 19, no. 4, pp. 521–528, Oct. 2013, doi: 10.1007/s12298-013-0186-2.
[7] R. F. Ratke et al., “Cashew gum hydrogel as an alternative to minimize the effect of drought stress on soybean,” Sci. Rep., vol. 14, no. 1, p. 2159, Jan. 2024, doi: 10.1038/s41598-024-52509-2.
[8] R. Hernández-Campos, C. Robles, and A. Calderín García, “EFECTO DE ÁCIDOS HÚMICOS EN EL CRECIMIENTO VEGETAL Y LA PROTECCIÓN CONTRA EL ESTRÉS HÍDRICO EN POBLACIONES SELECCIONADAS DE MAÍZ NATIVO DE MÉXICO,” Rev. Fitotec. Mex., vol. 44, no. 4, p. 561, Dec. 2021, doi: 10.35196/rfm.2021.4.561.
[9] F. Hellal, S. El Sayed, D. M. R. A. Basha, and H. H. A. Kader, “Mitigation of water stress by compost and arginine application and its impacts on barley production,” Bull. Natl. Res. Cent., vol. 48, no. 1, p. 25, Feb. 2024, doi: 10.1186/s42269-024-01178-2.
[10] U. K. Ghosh, Md. N. Islam, Md. N. Siddiqui, and Md. A. R. Khan, “Understanding the roles of osmolytes for acclimatizing plants to changing environment: a review of potential mechanism,” Plant Signal. Behav., vol. 16, no. 8, p. 1913306, Aug. 2021, doi: 10.1080/15592324.2021.1913306.
[11] D. Mehta and S. Vyas, “Comparative bio-accumulation of osmoprotectants in saline stress tolerating plants: A review,” Plant Stress, vol. 9, p. 100177, Sep. 2023, doi: 10.1016/j.stress.2023.100177.
[12] G. Forlani, K. S. Makarova, M. Ruszkowski, M. Bertazzini, and B. Nocek, “Evolution of plant δ1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives,” Front. Plant Sci., vol. 6, Aug. 2015, doi: 10.3389/fpls.2015.00567.
[13] K. Jamshidi Goharrizi, A. Baghizadeh, S. Karami, M. Nazari, and M. Afroushteh, “Expression of the W36, P5CS, P5CR, MAPK3, and MAPK6 genes and proline content in bread wheat genotypes under drought stress,” Cereal Res. Commun., vol. 51, no. 3, pp. 545–556, Sep. 2023, doi: 10.1007/s42976-022-00331-9.
[14] S. Hayat, Q. Hayat, M. N. Alyemeni, A. S. Wani, J. Pichtel, and A. Ahmad, “Role of proline under changing environments: A review,” Plant Signal. Behav., vol. 7, no. 11, pp. 1456–1466, Nov. 2012, doi: 10.4161/psb.21949.
[15] B. Jones et al., “Activation of proline biosynthesis is critical to maintain glutamate homeostasis during acute methamphetamine exposure,” Sci. Rep., vol. 11, no. 1, p. 1422, Jan. 2021, doi: 10.1038/s41598-020-80917-7.
[16] S. Lebreton, C. Cabassa-Hourton, A. Savouré, D. Funck, and G. Forlani, “Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities,” Front. Plant Sci., vol. 11, p. 602939, Dec. 2020, doi: 10.3389/fpls.2020.602939.
[17] X. Yang, M. Lu, Y. Wang, Y. Wang, Z. Liu, and S. Chen, “Response Mechanism of Plants to Drought Stress,” Horticulturae, vol. 7, no. 3, p. 50, Mar. 2021, doi: 10.3390/horticulturae7030050.
[18] D. Funck, L. Baumgarten, M. Stift, N. Von Wirén, and L. Schönemann, “Differential Contribution of P5CS Isoforms to Stress Tolerance in Arabidopsis,” Front. Plant Sci., vol. 11, p. 565134, Sep. 2020, doi: 10.3389/fpls.2020.565134.
[19] I. Pérez-Arellano, F. Carmona-Álvarez, J. Gallego, and J. Cervera, “Molecular Mechanisms Modulating Glutamate Kinase Activity. Identification of the Proline Feedback Inhibitor Binding Site,” J. Mol. Biol., vol. 404, no. 5, pp. 890–901, Dec. 2010, doi: 10.1016/j.jmb.2010.10.019.
[20] M. Hosseinifard, S. Stefaniak, M. Ghorbani Javid, E. Soltani, Ł. Wojtyla, and M. Garnczarska, “Contribution of Exogenous Proline to Abiotic Stresses Tolerance in Plants: A Review,” Int. J. Mol. Sci., vol. 23, no. 9, p. 5186, May 2022, doi: 10.3390/ijms23095186.
[21] C. Marco-Marín, F. Gil-Ortiz, I. Pérez-Arellano, J. Cervera, I. Fita, and V. Rubio, “A Novel Two-domain Architecture Within the Amino Acid Kinase Enzyme Family Revealed by the Crystal Structure of Escherichia coli Glutamate 5-kinase,” J. Mol. Biol., vol. 367, no. 5, pp. 1431–1446, Apr. 2007, doi: 10.1016/j.jmb.2007.01.073.
[22] T. Kaino, Y. Tasaka, Y. Tatehashi, and H. Takagi, “Functional Analysis of the C-Terminal Region of γ-Glutamyl Kinase of Saccharomyces cerevisiae,” Biosci. Biotechnol. Biochem., vol. 76, no. 3, pp. 454–461, Mar. 2012, doi: 10.1271/bbb.110682.
[23] Y. Guan et al., “Functional characterization of a gamma-glutamyl phosphate reductase ProA in proline biosynthesis and promoting expression of type three secretion system in Ralstonia solanacearum,” Front. Microbiol., vol. 13, p. 945831, Aug. 2022, doi: 10.3389/fmicb.2022.945831.
[24] R. Page et al., “Crystal structure of γ‐glutamyl phosphate reductase (TM0293) from Thermotoga maritima at 2.0 Å resolution,” Proteins Struct. Funct. Bioinforma., vol. 54, no. 1, pp. 157–161, Jan. 2004, doi: 10.1002/prot.10562.
[25] M. J. Iqbal, “Role of Osmolytes and Antioxidant Enzymes for Drought Tolerance in Wheat,” in Global Wheat Production, S. Fahad, A. Basir, and M. Adnan, Eds., InTech, 2018. doi: 10.5772/intechopen.75926.
[26] A. El Moukhtari, C. Cabassa-Hourton, M. Farissi, and A. Savouré, “How Does Proline Treatment Promote Salt Stress Tolerance During Crop Plant Development?,” Front. Plant Sci., vol. 11, p. 1127, Jul. 2020, doi: 10.3389/fpls.2020.01127.
| None |