scholarly journals Heterogeneous Expression of Cymbidium longibracteatum Magnesium Protoporphyrin IX Methyltransferase (ClChlM) Activates Chlorophyll Biosynthesis in Transgenic Tobacco

2021 ◽  
Vol 25 (04) ◽  
pp. 915-920
Author(s):  
Yu Jiang
Keyword(s):  
Binding Sites ◽  
Chloroplast Development ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Reading Frame ◽  
Putative Protein ◽  
Heterogeneous Expression ◽  
Localization Analysis ◽  
Substrate Binding Sites

Magnesium protoporphyrin IX methyltransferase (ChlM) plays an important role in the regulation of chlorophyll biosynthesis and chloroplast development. In the present study, we isolated a ChlM gene, designated ClChlM, from Cymbidium [Cymbidium longibracteatum (Wu & Chen) Chen & Liu]. The open reading frame (ORF) sequence of ClChlM was 945 bp and encoded a putative protein of 314 amino acids. The deduced ClChlM contained the conserved SAM/SAH binding pocket and substrate binding sites. Subcellular localization analysis of ClChlM revealed that the protein was localized in the chloroplast. Ectopic overexpression of ClChlM in tobacco (Nicotiana benthamiana Domin) increased ALA-synthesizing capacity and chlorophyll content and widely upregulated the expression level of photosynthesis-related genes, such as ClHemA, ClGSA, ClLhcb, ClCHLI, and ClCHLH. In conclusion, these results demonstrate that ClChlM plays a crucial role in the regulation of chlorophyll biosynthesis in C. longibracteatum and will help in breeding for leaf colour variance in the future. © 2021 Friends Science Publishers

scholarly journals One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP) 2 of Geobacillus stearothermophilus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Johanna Heuveling ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Content Type ◽  
Charged Amino Acids ◽  
Substrate Binding Sites ◽  
Positively Charged ◽  
Atp Binding Cassette

ABSTRACT ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN) 2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243–5248, 2015, https://doi.org/10.1073/pnas.1415037112 ). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP) 2 transporter of Geobacillus stearothermophilus , by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P] 2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer. IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus , encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

scholarly journals Characterization and Fine Mapping of a Yellow-Virescent Gene Regulating Chlorophyll Biosynthesis and Early Stage Chloroplast Development in Brassica napus

2020 ◽  
Vol 10 (9) ◽  
pp. 3201-3211 ◽  
Author(s):  
Chuanji Zhao ◽  
Lijiang Liu ◽  
Luqman Bin Safdar ◽  
Meili Xie ◽  
Xiaohui Cheng ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Chloroplast Development ◽  
Early Stage ◽  
Bulked Segregant Analysis ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Chloroplast Ultrastructure ◽  
Crop Species ◽  
Chlorophyll Accumulation ◽  
Nucleotide Mutation

Abstract Chlorophyll biosynthesis and chloroplast development are crucial to photosynthesis and plant growth, but their regulatory mechanism remains elusive in many crop species. We isolated a Brassica napus yellow-virescent leaf (yvl) mutant, which exhibited yellow-younger-leaf and virescent-older-leaf with decreased chlorophyll accumulation and delayed chloroplast development. We mapped yvl locus to a 70-kb interval between molecular markers yvl-O10 and InDel-O6 on chromosome A03 in BC2F2 population using whole genome re-sequencing and bulked segregant analysis. The mutant had a ‘C’ to ‘T’ substitution in the coding sequence of BnaA03.CHLH, which encodes putative H subunit of Mg-protoporphyrin IX chelatase (CHLH). The mutation resulted in an imperfect protein structure and reduced activity of CHLH. It also hampered the plastid encoded RNA polymerase which transcribes regulatory genes of photosystem II and I. Consequently, the chlorophyll a/b and carotenoid contents were reduced and the chloroplast ultrastructure was degraded in yvl mutant. These results explain that a single nucleotide mutation in BnaA03.CHLH impairs PEP activity to disrupt chloroplast development and chlorophyll biosynthesis in B. napus.

scholarly journals Differences in Photosynthesis of Variegated Temple Bamboo Leaves with Various Levels of Variegation are Related to Chlorophyll Biosynthesis and Chloroplast Development

2018 ◽  
Vol 143 (2) ◽  
pp. 144-153 ◽  
Author(s):  
Lingyan Chen ◽  
Jinli Lai ◽  
Tianyou He ◽  
Jundong Rong ◽  
Muhammad Waqqas Khan Tarin ◽  
...  
Keyword(s):  
Thylakoid Membrane ◽  
Chloroplast Development ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Light Response ◽  
Photosynthetic Parameters ◽  
Leaf Color ◽  
Light Response Curve ◽  
Bamboo Leaves ◽  
Series Of Experiments

Variegated temple bamboo (Sinobambusa tootsik f. luteoloalbostriata) is a species of ornamental bamboo (Bambusoideae) that has gained popularity because of its striped or variegated leaves. In this study, a series of experiments was conducted to determine the factors contributing to the leaf color of this species, which included the content of the photosynthetic pigments and the chlorophyll biosynthetic precursors, the photosynthetic parameters, and the microstructure and ultrastructure of the different phenotypes. Discoloration in the leaves of variegated temple bamboo plants is attributed to two possible pathways. One was a block in chlorophyll biosynthesis, which led to the failure in biosynthesis of the thylakoid membrane. The other one was a disruption in chloroplast development. The lack of thylakoid membrane may have inhibited the conversion of coproporphyrinogen III (Coprogen III) to protoporphyrin IX (Proto IX) during the chlorophyll biosynthesis because the enzyme responsible for this conversion, protogen oxidase, is bound to the thylakoid membrane. The abnormalities in chloroplasts and a low concentration of chlorophyll in the variegated leaves led to a significantly lower photosynthetic rate than in the entirely green leaves, as demonstrated in the light-response curve.

Substrate-binding sites in acetylcholinesterase

1991 ◽  
Vol 12 ◽  
pp. 422-426 ◽  
Author(s):  
Ferdinand Hucko ◽  
Jaak Järv ◽  
Christoph Weise
Keyword(s):  
Binding Sites ◽  
Substrate Binding ◽  
Substrate Binding Sites

scholarly journals Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

2005 ◽  
Vol 288 (2) ◽  
pp. F327-F333 ◽  
Author(s):  
Rémon A. M. H. Van Aubel ◽  
Pascal H. E. Smeets ◽  
Jeroen J. M. W. van den Heuvel ◽  
Frans G. M. Russel
Keyword(s):  
Binding Sites ◽  
Efflux Pump ◽  
Substrate Binding ◽  
Organic Anion ◽  
Apical Cell ◽  
Cell Uptake ◽  
Hek293 Cells ◽  
Hill Coefficient ◽  
Anion Transporter ◽  
Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

Sign in / Sign up

Export Citation Format

Share Document