Characterization and diverse evolution patterns of glycerol-3-phosphate dehydrogenase family genes in Dunaliella salina

Wu, Qian; Lan, Yanhong; Cao, Xiyue; Yao, Huiying; Qiao, Dan; Xu, Hui; Cao, Yi

doi:10.1016/j.gene.2019.05.056

Cited by 18 publications

(33 citation statements)

References 52 publications

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“…Glycerol-3-phosphate dehydrogenase (GPDH) is a rate-limiting enzyme in the glycerol synthesis pathway, and intracellular glycerol concentration functions as the counterbalancing osmolyte in D. salina [30]. Therefore, we further analysed the transcription levels of all DsGPDHs (DsGPDH1-7) [31], which were reported to be involved in glycerol synthesis. The expression profiles of these genes in the DsMEK1-X1-oe, DsMEK1-X2-oe, and DsMEK1-X2-RNAi lines and the control were analysed by qRT-PCR under salt stress (Fig 8).…”

Section: Dsmek1 Is Regulated By Alternative Splicingmentioning

confidence: 99%

“…For protein localization observation, DsMEK1-X1 and DsMEK1-X2 were inserted into the p1300-GFP expression vector using gene specific primers (primers No. 5-6 in Table S1) [31]. To transiently express the fusion proteins in protoplasts isolated from Arabidopsis leaves, PEG-mediated transformation was used to transfect the protoplasts with each DsMEK1::GFP construct [50].…”

Section: Subcellular Localizationmentioning

confidence: 99%

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Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Zhong

Cao

Zhang

et al. 2020

Preprint

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Background: Dunaliella salina can produce glycerol under salt stress, and this production can quickly adapt to changes in external salt concentration. Notably, glycerol is an ideal energy source. In recent years, it has been reported that the mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is highly important to study the relationship between the MAPK cascade pathway and salt stress in D. salina and modify it to increase the prodution of glycerol. Results: In our study, we identified and analysed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1 and DsMEK1-X2 were both localized in the cytoplasm. qRT-PCR assays showed that DsMEK1-X2 was induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol production compared to the control and DsMEK1-X1-oe under salt stress. Under salt stress, the expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control. This finding indicated that DsMEK1-X2 was involved in the regulation of DsGPDH expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increased the proline content and reduced the MDA content under salt stress, and DsMEK1-X1 was able to regulate oxidative stress; thus, we hypothesized that DsMEK1-X1 could reduce oxidative damage under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 could interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1; however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines did not exhibit increased gene expression, except for DsMAPKKK9. Conclusion: Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can respond to salt stress by two different pathways. The DsMEK1-X1 response to salt stress reduces oxidative damage; however, the DsMAPKKK1/2/3/9/10/17- DsMEK1-X2-DsMAPK1 cascade is involved in the regulation of DsGPDH expression and thus glycerol synthesis under salt stress.

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Section: Dsmek1 Is Regulated By Alternative Splicingmentioning

confidence: 99%

Section: Subcellular Localizationmentioning

confidence: 99%

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Zhong

Cao

Zhang

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Glycerol-3-phosphate dehydrogenase (GPDH) is a rate-limiting enzyme in the glycerol synthesis pathway and intracellular glycerol concentration functions as the counterbalancing osmolyte in D. salina [37]. So we further analyzed the transcription levels of all DsGPDH (DsGPDH1-7) [38], which were reported to be involved in glycerol synthesis. The expression profile of these genes in the DsMEK1-X1-oe, DsMEK1-X2-oe, DsMEK1-X2-RNAi lines and control were analyzed by qRT-PCR under salt stress (Fig.…”

Section: Dsmek1 Is Regulated By Alternative Splicingmentioning

confidence: 99%

“…For protein localization observation, DsMEK1-X1 and DsMEK1-X2 were inserted into the p1300-GFP expression vector using gene specific primers (primers No. 3-4 in Table S1) [38]. To transiently express the fusion proteins in protoplasts isolated from Arabidopsis leaves, PEG-mediated transformation was used to transfect the protoplasts with each DsMEK1::GFP construct.…”

Section: Subcellular Localizationmentioning

confidence: 99%

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) is involved different way in salt stress regulation in Dunaliella salina

Zhong

Cao

Zhang

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background：Dunaliella salina can produce a large amount of glycerol under salt stress, which can quickly adapt to the change of external salt concentration, and glycerol is one of the ideal energy sources. In recent years, it has been reported that Mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is urgent to study the relationship between MAPK cascade pathway and salt stress in D. salina, and help it to increase the content of glycerol. Results： In our study, we identified and analyzed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1, DsMEK1-X2 both localized in the cytoplasm. The qRT-PCR assays showed that DsMEK1-X2 induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol compared to the control and DsMEK1-X1-oe under salt stress. The expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control under salt stress. It means that DsMEK1-X2 is involved in the regulation of DsGPDHs expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increasing the proline content and reducing the MDA content under salt stress, and DsMEK1-X1 can regulate oxidative stress, thus we speculate that DsMEK1-X1 can reduce the damage of oxidative under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 can interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1, however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines do not increased genes expression, except for DsMAPKKK9. Conclusion： Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can response to salt stress in two different pathways, DsMEK1-X1 response to salt stress by reducing oxidative damage, however, DsMAPKKK1/2/3/9/10/17- DsMEK1-X2-DsMAPK1 cascade is involved in the regulated of DsGPDH expression and thus glycerol synthesis under salt stress.

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“…Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme, 61 and has therefore been a focus of study in both the contexts of for example lipid biosynthesis, 62,63 thermogenesis, 64,65 hepatic steatosis, 66 and organismal responses to abiotic stress. 48,[67][68][69][70][71] There have also been a range of structural analyses of GPDHs from different organisms that provide important information into the overall structure of the enzyme 5,11,72 (including characterizing the flexible loop 292-LNGQKL-297), as well as biochemical and kinetic studies of the catalytic activity of different GPDHs, 1,5,11,73 and the activation of GPDH by phosphate dianions, 9, 74 using a similar substrate-in-pieces approach as applied to other enzymes such as TIM 13,14 and OMPDC. 15 In addition, examination of primary deuterium kinetic isotope effects (1° DKIEs) of the hydride transfer catalyzed by wild-type and substituted GPDHs show that these values fall within a very narrow range (2.4-3.1) for reactions spanning a 9.1 kcal•mol -1 change in reaction driving force (∆∆G ‡ ).…”

Section: Identifying the Mechanism Of The Reduction Of Dhap By Wild-tmentioning

confidence: 99%

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

Mhashal¹,

Romero‐Rivera²,

Mydy³

et al. 2020

Preprint

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<div> <div> <div> <p>Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend the experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a novel crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted variants of hlGPDH and investigated the effect of mutations on the catalysis from a detailed structural study. Our structural data and simulations also illustrate the critical role of the R269 side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297), thus rationalizing a tremendous 41,000-fold decrease experimentally in the turnover number, kcat, upon truncating this residue. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of novel allosteric inhibitors targeting this biomedically important enzyme.</p></div></div></div>

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Characterization and diverse evolution patterns of glycerol-3-phosphate dehydrogenase family genes in Dunaliella salina

Cited by 18 publications

References 52 publications

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) is involved different way in salt stress regulation in Dunaliella salina

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

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