TY  - JOUR
A1  - Ritte, Gerhard
A1  - Heydenreich, Matthias
A1  - Mahlow, Sebastian
A1  - Haebel, Sophie
A1  - Koetting, Oliver
A1  - Steup, Martin
T1  - Phosphorylation of C6- and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases
JF  - FEBS letters : the journal for rapid publication of short reports in molecular biosciences
N2  - Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wildtype plants and mutants lacking either GWD or PWD using P-31 NMR. Phosphorylation at both C6- and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid-hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively.
KW  - starch phosphorylation
KW  - GWD
KW  - PWD
KW  - P-31 NMR
Y1  - 2006
U6  - https://doi.org/10.1016/j.febslet.2006.07.085
SN  - 0014-5793
VL  - 580
IS  - 20
SP  - 4872
EP  - 4876
PB  - Elsevier
CY  - Amsterdam
ER  - 
TY  - JOUR
A1  - Malinova, Irina
A1  - Mahto, Harendra
A1  - Brandt, Felix
A1  - AL-Rawi, Shadha
A1  - Qasim, Hadeel
A1  - Brust, Henrike
A1  - Hejazi, Mahdi
A1  - Fettke, Jörg
T1  - EARLY STARVATION1 specifically affects the phosphorylation action of starch-related dikinases
JF  - The plant journal
N2  - Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various invitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, -glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.
KW  - Arabidopsis thaliana
KW  - EARLY STARVATION1
KW  - glucan
KW  - phosphoglucan
KW  - starch granule surface
KW  - starch phosphorylation
KW  - water dikinase
Y1  - 2018
U6  - https://doi.org/10.1111/tpj.13937
SN  - 0960-7412
SN  - 1365-313X
VL  - 95
IS  - 1
SP  - 126
EP  - 137
PB  - Wiley
CY  - Hoboken
ER  - 
TY  - THES
A1  - Mahto, Harendra
T1  - In vitro analysis of Early Starvation 1 (ESV1) and Like Early Starvation 1 (LESV) on starch degradation with focus on glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD)
N2  - Starch is an insoluble polyglucan, comprises of two polymers, namely, the branched α-1,4: α-1,6-D-glucan amylopectin and the almost unbranched α-1,4-D-glucan amylose. The growth of all plants is directly dependent on the accumulation of transitory starch during the daytime when photosynthesis takes place and subsequently starch degradation during the night. Starch phosphorylation takes place by starch-related dikinases called α-glucan, water dikinase (GWD), and phosphoglucan, water dikinase (PWD), and is a very important step in starch degradation. The biochemical mechanisms of phosphorylation of starch are not properly understood. Recent studies have found that there are two starch binding proteins namely, Early Starvation1 (ESV1) and Like Early Starvation1 (LESV), which play an important role in starch metabolism. It has been shown that ESV1 and LESV proteins affect the starch phosphorylation activity of GWD and PWD enzymes, which control the rate of degradation of starch granules. In this thesis, various in vitro assays were performed to identify and understand the mechanism of recombinant proteins; ESV1 and LESV on the starch degradation. The starch degradation was performed by phosphorylation enzymes, GWD and PWD separately. In various enzymatic assays, the influence of the ESV1 and LESV on the actions of GWD and PWD on the surfaces of different native starch granules were analysed. Furthermore, ESV1 and LESV have specifically shown influences on the phosphorylation activities of GWD and PWD on the starch granule surfaces in an antagonistic pattern in such a way that, the GWD mediated phosphorylation were significantly reduced while PWD mediated phosphorylation were significantly increased respectively. In another set of experiments, ISA and BAM hydrolyzing enzymes were used to alter the structure of starch, and then determine the effect of both dikinases mediated phosphorylation in the presence of ESV1 and LESV on the altered starch granules surfaces. In these results, significant decreases in both GWD and PWD mediated phosphorylation were observed in all the treatments containing either ESV1 or LESV proteins only or both ESV1 and LESV. It was also found that LESV preferentially binds to both amylose and amylopectin, while ESV1 binds to highly ordered glucans such as maltodextrins and amylopectin, which are crystalline in structure. Both ESV1 or LESV proteins either individually or in combination have shown influence on the activity of GWD and PWD phosphate incorporation into the starch granules via reduction even though at different percentages depending on the sources of starch, therefore it is difficult to distinguish the specific function between them. The biochemical studies have shown that protein-glucan interaction specifically between ESV1 or LESV or in combination with different species of starch granules has very strong surface binding, or it might be possible that both the proteins not only bind to the surface of the starch granules but also have entered deep inside the glucan structure of the starch granules. However, the results also revealed that ESV1 and LESV did not alter the autophosphorylation of the dikinases. Also, the chain length distribution pattern of the released glucan chains after treatment of starch with ISA enzyme was evaluated with respect to the degree of polymerization (DP) of the different starch granules. Capillary electrophoresis was employed to study the effect of LESV and ESV1 on the chain length distribution. In summary, this study confirms that ESV1 and LESV play an important role in organizing and regulating the starch metabolism process. In the later half, studies were performed to monitor whether the metabolism of carbohydrates and partitioning, contribute to the higher salt tolerance of the facultative halophyte Hordeum marinum when compared to glycophyte Hordeum vulgare. Seedlings with the same size from both species were hydroponically grown at 0, 150, and 300 mM of NaCl for 3 weeks. H. marinum maintained a high relative growth rate, which was found concomitant in higher aptitude plants to maintain efficient shoot tissue hydration and integrity of membrane under salt conditions when compared to H. vulgare. Hence, our data suggested that the change in the starch storage, distribution of soluble sugar concentrations between source and sink organs, and also changes in the level of enzymes involved in the starch metabolism was significant to give insights into the importance of carbohydrate metabolism in barley species with regards to the salt tolerance. Although these results are still in their nascent state, it could be vital for other researchers to formulate future studies. The preliminary results which were studies about the carbohydrate metabolism and partitioning in salt responses in the halophyte H. marinum and the glycophyte H. vulgare revealed that salt tolerance in barley species is not due to osmotic adjustments, but due to other reasons that were not explored in the past studies. However, the activity of DPE2 in H. vulgare was not hampered by the presence of NaCl as observed. While Pho1 and Pho2, activities were highly increased in cultivated barley. These findings could be suggestive of a possible role of these enzymes in the responses of carbohydrate metabolism to salinity. When sea and cultivated barley species were compared, it was discovered that the former had more versatility in carbohydrate metabolism and distribution.
N2  - Stärke ist ein unlösliches Polyglucan, das aus zwei Polymeren besteht, nämlich dem verzweigten α-1,4: α-1,6-D-Glucan Amylopektin und dem fast unverzweigten α-1,4-D-Glucan Amylose. Das Wachstum aller Pflanzen hängt direkt von der Akkumulation transitorischer Stärke während des Tages, wenn die Photosynthese stattfindet, und dem anschließenden Stärkeabbau während der Nacht ab. Die Phosphorylierung von Stärke erfolgt durch stärkeverwandte Dikinasen, die α-Glucan-Wasser-Dikinase (GWD) und Phosphoglucan-Wasser-Dikinase (PWD), und ist ein entscheidender Schritt beim Stärkeabbau. Die biochemischen Mechanismen der Phosphorylierung von Stärke sind nicht genau bekannt. Jüngste Studien haben ergeben, dass es zwei stärkebindende Proteine gibt, nämlich Early Starvation1 (ESV1) und Like Early Starvation1 (LESV), die eine wichtige Rolle im Stärkestoffwechsel spielen. Es hat sich gezeigt, dass ESV1- und LESV-Proteine die Stärkephosphorylierungsaktivität der GWD- und PWD-Enzyme beeinflussen, die die Geschwindigkeit des Abbaus von Stärkekörnern steuern. In dieser Arbeit wurden verschiedene In-vitro-Tests durchgeführt, um den Mechanismus der rekombinanten Proteine ESV1 und LESV auf den Stärkeabbau zu identifizieren und zu verstehen.Der Stärkeabbau wurde von den Phosphorylierungsenzymen GWD und PWD getrennt durchgeführt. In verschiedenen enzymatischen Assays wurde der Einfluss von ESV1 und LESV auf die Wirkung von GWD und PWD auf die Oberflächen verschiedener nativer Stärkekörner analysiert. Darüber hinaus haben ESV1 und LESV spezifisch Einflüsse auf die Phosphorylierungsaktivitäten von GWD und PWD auf den Oberflächen der Stärkekörner in einem antagonistischen Muster gezeigt, so dass die GWD-vermittelte Phosphorylierung signifikant reduziert wurde, während die PWD-vermittelte Phosphorylierung signifikant erhöht wurde. In einer anderen Versuchsreihe wurden ISA- und BAM verwendet, um die Struktur der Stärke zu verändern und dann die Auswirkungen der durch beide Dikinasen vermittelten Phosphorylierung in Gegenwart von ESV1 und LESV auf die veränderten Oberflächen der Stärkekörner zu bestimmen. In diesen Ergebnissen wurde ein signifikanter Rückgang der GWD- und PWD-vermittelten Phosphorylierung in allen Behandlungen beobachtet, die entweder nur ESV1- oder LESV-Proteine oder sowohl ESV1 als auch LESV enthielten. Es wurde auch festgestellt, dass LESV vorzugsweise an Amylose und Amylopektin bindet, während ESV1 an hochgeordnete Glucane wie Maltodextrine und Amylopektin bindet, die eine kristalline Struktur aufweisen. Sowohl ESV1- als auch LESV-Proteine haben entweder einzeln oder in Kombination einen Einfluss auf die Aktivität des GWD- und PWD-Phosphateinbaus in die Stärkekörner durch Reduktion gezeigt, jedoch zu unterschiedlichen Prozentsätzen, je nach Stärkequelle, so dass es schwierig ist, ihre spezifische Funktion zu unterscheiden. Die biochemischen Untersuchungen zeigen, dass die Protein-Glucan-Interaktion speziell zwischen ESV1 oder LESV oder in Kombination mit verschiedenen Arten von Stärkekörnern eine sehr starke Oberflächenbindung aufweist, oder es ist möglich, dass beide Proteine nicht nur an die Oberfläche der Stärkekörner binden, sondern auch tief in die Glucanstruktur der Stärkekörner eingedrungen sind. Die Ergebnisse zeigten jedoch auch, dass ESV1 und LESV die Autophosphorylierung der Dikinasen nicht veränderten. Außerdem wurde die Kettenlängenverteilung der freigesetzten Glucanketten nach Behandlung der Stärke mit dem ISA-Enzym im Hinblick auf den Polymerisationsgrad (DP) der verschiedenen Stärkekörner bewertet. Mit Hilfe der Kapillarelektrophorese wurde die Wirkung von LESV und ESV1 auf die Kettenlängenverteilung untersucht. Zusammenfassend bestätigt diese Studie, dass ESV1 und LESV eine wichtige Rolle bei der Organisation und Regulierung des Stärkestoffwechsels spielen. In der zweiten Hälfte wurden Untersuchungen durchgeführt, um zu prüfen, ob der Stoffwechsel von Kohlenhydraten und deren Verteilung zu der höheren Salztoleranz des fakultativen Halophyten Hordeum marinum im Vergleich zum Glykophyten Hordeum vulgare beitragen. Die gleich großen Sämlinge beider Arten wurden 3 Wochen lang bei 0, 150 und 300 mM NaCl hydroponisch gezogen. H. marinum wies eine hohe relative Wachstumsrate auf, die mit einer höheren Fähigkeit der Pflanzen einherging, unter Salzbedingungen eine effiziente Hydratation des Sprossgewebes und die Integrität der Membran aufrechtzuerhalten, als dies bei H. vulgare der Fall war. Unsere Daten deuten also darauf hin, dass die Veränderungen in der Stärkespeicherung, die Verteilung der Konzentrationen löslicher Zucker zwischen Source- und Sinkorganen und auch die Veränderungen in der Menge der am Stärkestoffwechsel beteiligten Enzyme von Bedeutung sind und Einblicke in die Bedeutung des Kohlenhydratstoffwechsels bei Gerstenarten im Hinblick auf die Salztoleranz geben. Obwohl sich diese Ergebnisse noch im Anfangsstadium befinden, könnten sie für andere Forscher bei der Formulierung künftiger Studien von entscheidender Bedeutung sein. Die vorläufigen Ergebnisse der Studien über den Kohlenhydratstoffwechsel und die Verteilung der Kohlenhydrate bei Salzreaktionen im Halophyten H. marinum und im Glykophyten H. vulgare haben gezeigt, dass die Salztoleranz bei Gerstenarten nicht auf osmotische Anpassungen zurückzuführen ist, sondern auf andere Gründe, die in den bisherigen Studien nicht untersucht wurden. Die Aktivität von DPE2 in H. vulgare wurde jedoch nicht wie beobachtet durch die Anwesenheit von NaCl beeinträchtigt. Dagegen waren die Aktivitäten von Pho1 und Pho2 in kultivierter Gerste stark erhöht. Diese Ergebnisse könnten auf eine mögliche Rolle dieser Enzyme bei der Reaktion des Kohlenhydratstoffwechsels auf den Salzgehalt hinweisen. Beim Vergleich von Meeres- und Kulturgerstenarten wurde festgestellt, dass erstere eine größere Vielseitigkeit im Kohlenhydratstoffwechsel und in der Kohlenhydratverteilung aufweisen.
KW  - Arabidopsis thaliana
KW  - starch phosphorylation
KW  - phosphoglucan
KW  - starch granule surface
KW  - Early Starvation 1
Y1  - 2022
ER  - 
TY  - JOUR
A1  - Mahlow, Sebastian
A1  - Hejazi, Mahdi
A1  - Kuhnert, Franziska
A1  - Garz, Andreas
A1  - Brust, Henrike
A1  - Baumann, Otto
A1  - Fettke, Jörg
T1  - Phosphorylation of transitory starch by -glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules
JF  - New phytologist : international journal of plant science
N2  - Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, -amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.
KW  - Arabidopsis thaliana
KW  - glucan
KW  - water dikinase (GWD)
KW  - sex1-8
KW  - starch granule surface
KW  - starch phosphorylation
Y1  - 2014
U6  - https://doi.org/10.1111/nph.12801
SN  - 0028-646X
SN  - 1469-8137
VL  - 203
IS  - 2
SP  - 495
EP  - 507
PB  - Wiley-Blackwell
CY  - Hoboken
ER  - 
TY  - JOUR
A1  - Hejazi, Mahdi
A1  - Steup, Martin
A1  - Fettke, Jörg
T1  - The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions
JF  - The FEBS journal
N2  - The plant genome encodes at least two distinct and evolutionary conserved plastidial starch-related dikinases that phosphorylate a low percentage of glucosyl residues at the starch granule surface. Esterification of starch favours the transition of highly ordered a-glucans to a less ordered state and thereby facilitates the cleavage of interglucose bonds by hydrolases. Metabolically most important is the phosphorylation at position C6, which is catalysed by the glucan, water dikinase (GWD). The reactions mediated by recombinant wild-type GWD from Arabidopsis thaliana (AtGWD) and from Solanum tuberosum (StGWD) were studied. Two mutated proteins lacking the conserved histidine residue that is indispensible for glucan phosphorylation were also included. The wild-type GWDs consume approximately 20% more ATP than is required for glucan phosphorylation. Similarly, although incapable of phosphorylating a-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of a-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [gamma-33P]ATP, the consumption of ATP includes the transfer of the gamma-phosphate group to the GWD protein but this autophosphorylation does not require the conserved histidine residue. Thus, the GWD proteins possess two vicinal phosphorylation sites, both of which are transiently phosphorylated. Following autophosphorylation at both sites, native dikinases flexibly use various terminal phosphate acceptors, such as water, alpha-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors.
KW  - glucan phosphorylation
KW  - glucan
KW  - water dikinase
KW  - protein autophosphorylation
KW  - starch metabolism
KW  - starch phosphorylation
Y1  - 2012
U6  - https://doi.org/10.1111/j.1742-4658.2012.08576.x
SN  - 1742-464X
VL  - 279
IS  - 11
SP  - 1953
EP  - 1966
PB  - Wiley-Blackwell
CY  - Malden
ER  -