scholarly journals Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

2004 ◽  
Vol 16 (3) ◽  
pp. 694-708 ◽  
Author(s):  
Ian J. Tetlow ◽  
Robin Wait ◽  
Zhenxiao Lu ◽  
Rut Akkasaeng ◽  
Caroline G. Bowsher ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Phosphorylation ◽  
Protein Interactions ◽  
Branching Enzyme ◽  
Protein Protein Interactions ◽  
Starch Branching Enzyme

Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules

2012 ◽  
Vol 448 (3) ◽  
pp. 373-387 ◽  
Author(s):  
Fushan Liu ◽  
Nadya Romanova ◽  
Elizabeth A. Lee ◽  
Regina Ahmed ◽  
Martin Evans ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Interactions ◽  
Starch Granule ◽  
Protein Complexes ◽  
Starch Synthase ◽  
Starch Granules ◽  
Branching Enzyme ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Starch Branching Enzyme

The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein–protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein–protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.

Production of High Amylose and Resistant Starch Rice Through Targeted Mutagenesis of Starch Branching Enzyme Iib by Crispr/cas9

2021 ◽  
Author(s):  
Hsi-Chao Wang ◽  
Yu-Chia Hsu ◽  
Yong-Pei Wu ◽  
Su-Ying Yeh ◽  
Maurice S. B. Ku
Keyword(s):  
Gene Expression ◽  
Enzyme Activity ◽  
Resistant Starch ◽  
Targeted Mutagenesis ◽  
Rice Starch ◽  
Branching Enzyme ◽  
Starch Branching Enzyme ◽  
Homozygous Mutant ◽  
Mutant Lines ◽  
High Amylose

Abstract Rice is the staple food for half of the world’s population. Starch accounts for 80-90% of the total mass of rice seeds, and rice starch is low in resistant starch (RS) with a high glycemic index (GI). RS has gained important since it is beneficial in preventing various diseases. Starch branching enzyme IIb (SBEIIb) plays a key role in the amylopectin synthesis pathway in the endosperm of cereals. Down-regulation of SBEIIb in several important crops has led to high amylose, high RS and low GI starch. In this study, we mutated OsSBEIIb in the japonica rice cultivar TNG82 through CRISPR/Cas9 and investigated the molecular and physicochemical modifications in OsSBEIIb mutant lines, e.g., gene expression, enzyme activity, apparent amylose content (AAC), RS and GI. As expected, the levels of modification in these starch related traits in heterozygous mutant lines were about half as those of homozygous mutant lines. Gene expression and enzyme activity of OsSBEIIb were down-regulated significantly while AAC and RS contents increased progressively from 17.4% and 0.5% in WT, respectively, to as high as 25.0% and 7.5% in heterozygous mutant lines and 36.0% and 12.0% in homozygous mutant lines. Consequently, with increased RS and decreased rate of reducing sugar production, GI progressively decreased in heterozygous and homozygous mutant rice endosperms by 11% and 28%, respectively. Our results demonstrate that it has huge potential for precise and efficient generation of high RS and low GI rice through CRISPR/Cas9 to provide a more suitable source of starch for type II diabetes.

scholarly journals The inner mechanics of Rhodopsin Guanylyl Cyclase during 2cGMP-formation revealed by real-time FTIR spectroscopy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Paul Fischer ◽  
Shatanik Mukherjee ◽  
Enrico Peter ◽  
Matthias Broser ◽  
Franz Bartl ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Real Time ◽  
Protein Interactions ◽  
Uv Light ◽  
Spectroscopic Characterization ◽  
Binding Pocket ◽  
Molecular Signaling ◽  
Protein Protein Interactions ◽  
Regulatory Influence ◽  
Truncated Variants

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

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