scholarly journals Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively

1999 ◽  
Vol 340 (1) ◽  
pp. 183-191 ◽  
Author(s):  
Kay DENYER ◽  
Darren WAITE ◽  
Saddik MOTAWIA ◽  
Birger Lindberg MØLLER ◽  
Alison M. SMITH
Keyword(s):  
Starch Synthase ◽  
Kinetic Properties ◽  
Starch Granules ◽  
Wild Type ◽  
Lower Affinity ◽  
Functional Differences ◽  
Glucose Molecule ◽  
Granule Bound Starch Synthase

Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed.

Multiple effects of the starch synthase II mutation in developing wheat endosperm

2007 ◽  
Vol 34 (5) ◽  
pp. 431 ◽  
Author(s):  
Behjat Kosar-Hashemi ◽  
Zhongyi Li ◽  
Oscar Larroque ◽  
Ahmed Regina ◽  
Makoto Yamamori ◽  
...  
Keyword(s):  
Triticum Aestivum L ◽  
Endosperm Development ◽  
Starch Synthase ◽  
Starch Granules ◽  
Branching Enzyme ◽  
Wild Type ◽  
Drastic Reduction ◽  
Branching Patterns ◽  
Soluble Phase ◽  
Mutant Lines

A line of wheat (Triticum aestivum L.), sgp-1, that does not express starch synthase II (SSII, also known as SGP-1) has previously been reported. In this study, F1 derived doubled haploid lines with homozygous wild type or mutant alleles for SGP-1 genes were identified from a cross between the original mutant and a wild type Australian cultivar. Analysis of the starch granules showed that in the mutant lines they are markedly distorted from 15 days postanthesis during grain development. Starch branching patterns showed an increase in the proportion of short chains (DP 6–10) at an earlier stage, but this increase became much more pronounced at 15 days postanthesis and persisted until maturity. There was also a consistent and drastic reduction throughout seed development in the relative amounts of starch branching enzyme II (SBEII, comprising SBEIIa and SBEIIb) and starch synthase I (SSI) bound to the starch granules. In the soluble phase, however, there was relatively little change in the amount of SBEIIb, SBEIIa or SSI protein. Therefore loss of SSII specifically leads to the loss of SBEIIb, SBEIIa and SSI protein in the granule-bound phase and the effect of this mutation is clearly manifest from the mid-stage of endosperm development in wheat.

Gli1can rescue the in vivo function ofGli2

Development ◽  
2001 ◽  
Vol 128 (24) ◽  
pp. 5161-5172 ◽  
Author(s):  
Chunyang Brian Bai ◽  
Alexandra L. Joyner
Keyword(s):  
Spinal Cord ◽  
Sonic Hedgehog ◽  
Target Genes ◽  
Wild Type ◽  
Shh Signaling ◽  
Gli Proteins ◽  
Functional Differences ◽  
Expression Studies ◽  
Null Mutants

In mice, three Gli genes are thought to mediate sonic hedgehog (Shh) signaling collectively. Mis-expression studies and analysis of null mutants for each gene have indicated that the Gli proteins have different functions. In particular, Gli1 appears to be a constitutive activator, and Gli2 and Gli3 have repressor functions. To determine the precise functional differences between Gli1 and Gli2, we have expressed Gli1 in place of Gli2 from the endogenous Gli2 locus in mice. Strikingly, a low level of Gli1 can rescue all the Shh signaling defects in Gli2 mutants; however, only in the presence of a wild-type Shh gene. These studies demonstrate that only the activator function of Gli2 is actually required, and indicates that in specific situations, Shh can modulate the ability of Gli1 to activate target genes. Furthermore, expression of both copies of Gli1 in place of Gli2 does not disrupt spinal cord patterning, but does result in new gain-of-function defects that lead to lethality. We show that the defects are enhanced when Gli3 function is reduced, demonstrating that an important difference between Gli1 and Gli2 is the ability of Gli1 to antagonize Gli3 function.

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.

scholarly journals PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

PLoS Biology ◽  
2015 ◽  
Vol 13 (2) ◽  
pp. e1002080 ◽  
Author(s):  
David Seung ◽  
Sebastian Soyk ◽  
Mario Coiro ◽  
Benjamin A. Maier ◽  
Simona Eicke ◽  
...  
Keyword(s):  
Protein Targeting ◽  
Starch Synthase ◽  
Starch Granules ◽  
Granule Bound Starch Synthase

scholarly journals Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of Lactococcus lactis

2000 ◽  
Vol 182 (6) ◽  
pp. 1600-1608 ◽  
Author(s):  
Antonia Picon ◽  
Edmund R. S. Kunji ◽  
Frank C. Lanfermeijer ◽  
Wil N. Konings ◽  
Bert Poolman
Keyword(s):  
Lactococcus Lactis ◽  
Fold Increase ◽  
Binding Activity ◽  
Kinetic Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Serovar Typhimurium ◽  
Peptide Binding Site ◽  
Oligopeptide Transport

ABSTRACT The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppAgene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA fromSalmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similarKm for transport, whereas theV max was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

scholarly journals Conserved residues in Ycf54 are required for protochlorophyllide formation in Synechocystis sp. PCC 6803

2017 ◽  
Vol 474 (5) ◽  
pp. 667-681 ◽  
Author(s):  
Sarah Hollingshead ◽  
Sophie Bliss ◽  
Patrick J. Baker ◽  
C. Neil Hunter
Keyword(s):  
Hydrogen Bonding ◽  
Protoporphyrin Ix ◽  
Wild Type ◽  
Structural Effects ◽  
Conserved Residues ◽  
Functional Differences ◽  
In Vivo Effects ◽  
Mgpme Cyclase ◽  
Synechocystis Sp

Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME–cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME–cyclase activity in the cyanobacterium Synechocystis. Synechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME–cyclase step, whereas the A9G mutation reduced Ycf54 levels by ∼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by ∼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME–cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.

scholarly journals Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity

BIO-PROTOCOL ◽  
2014 ◽  
Vol 4 (23) ◽  
Author(s):  
Tomás Albi ◽  
M. Ortiz-Marchena ◽  
M. Ruiz ◽  
José Romero ◽  
Federico Valverde
Keyword(s):  
Starch Synthase ◽  
Starch Granules ◽  
Granule Bound Starch Synthase

scholarly journals Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides

1999 ◽  
Vol 342 (3) ◽  
pp. 647-653 ◽  
Author(s):  
Kay DENYER ◽  
Darren WAITE ◽  
Anne EDWARDS ◽  
Cathie MARTIN ◽  
Alison M. SMITH
Keyword(s):  
Starch Granule ◽  
Specific Interaction ◽  
Unique Property ◽  
Starch Synthase ◽  
The Other ◽  
Soluble Form ◽  
The Matrix ◽  
Granule Bound Starch Synthase

This paper examines the properties in soluble form of two isoforms of starch synthase. One of these, granule-bound starch synthase I (GBSSI), is responsible for the synthesis of amylose inside the amylopectin matrix of the starch granule in vivo. The other, starch synthase II (SSII), is involved in amylopectin synthesis. Both isoforms can use amylopectin and malto-oligosaccharide as substrates in vitro. As well as acting as a substrate for GBSSI, amylopectin acts as an effector of this isoform, increasing the rate at which it elongates malto-oligosaccharides and promoting a processive rather than distributive mode of elongation of these compounds. The affinity of GBSSI for amylopectin as an effector is greater than its affinity for amylopectin as a substrate. The rate and mode of elongation of malto-oligosaccharides by SSII are not influenced by amylopectin. These results suggest that specific interaction with amylopectin in the matrix of the starch granule is a unique property of GBSSI and is critical in determining the nature of its products.

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