Shadha AL-Rawi

• Depending on the biochemical and biotechnical approach, the aim of this work was to understand the mechanism of protein-glucan interactions in regulation and control of starch degradation. Although starch degradation starts with the phosphorylation process, the mechanisms by which this process is controlling and adjusting starch degradation are not yet fully understood. Phosphorylation is a major process performed by the two dikinases enzymes α-glucan, water dikinase (GWD) and phosphoglucan water dikinase (PWD). GWD and PWD enzymes phosphorylate the starch granule surface; thereby stimulate starch degradation by hydrolytic enzymes. Despite these important roles for GWD and PWD, so far the biochemical processes by which these enzymes are able to regulate and adjust the rate of phosphate incorporation into starch during the degradation process haven‘t been understood. Recently, some proteins were found associated with the starch granule. Two of these proteins are named Early Starvation Protein 1 (ESV1) and its homologue Like-EarlyDepending on the biochemical and biotechnical approach, the aim of this work was to understand the mechanism of protein-glucan interactions in regulation and control of starch degradation. Although starch degradation starts with the phosphorylation process, the mechanisms by which this process is controlling and adjusting starch degradation are not yet fully understood. Phosphorylation is a major process performed by the two dikinases enzymes α-glucan, water dikinase (GWD) and phosphoglucan water dikinase (PWD). GWD and PWD enzymes phosphorylate the starch granule surface; thereby stimulate starch degradation by hydrolytic enzymes. Despite these important roles for GWD and PWD, so far the biochemical processes by which these enzymes are able to regulate and adjust the rate of phosphate incorporation into starch during the degradation process haven‘t been understood. Recently, some proteins were found associated with the starch granule. Two of these proteins are named Early Starvation Protein 1 (ESV1) and its homologue Like-Early Starvation Protein 1 (LESV). It was supposed that both are involved in the control of starch degradation, but their function has not been clearly known until now. To understand how ESV1 and LESV-glucan interactions are regulated and affect the starch breakdown, it was analyzed the influence of ESV1 and LESV proteins on the phosphorylating enzyme GWD and PWD and hydrolysing enzymes ISA, BAM, and AMY. However, the analysis determined the location of LESV and ESV1 in the chloroplast stroma of Arabidopsis. Mass spectrometry data predicted ESV1and LESV proteins as a product of the At1g42430 and At3g55760 genes with a predicted mass of ~50 kDa and ~66 kDa, respectively. The ChloroP program predicted that ESV1 lacks the chloroplast transit peptide, but it predicted the first 56 amino acids N-terminal region as a chloroplast transit peptide for LESV. Usually, the transit peptide is processed during transport of the proteins into plastids. Given that this processing is critical, two forms of each ESV1 and LESV were generated and purified, a full-length form and a truncated form that lacks the transit peptide, namely, (ESV1and tESV1) and (LESV and tLESV), respectively. Both protein forms were included in the analysis assays, but only slight differences in glucan binding and protein action between ESV1 and tESV1 were observed, while no differences in the glucan binding and effect on the GWD and PWD action were observed between LESV and tLESV. The results revealed that the presence of the N-terminal is not massively altering the action of ESV1 or LESV. Therefore, it was only used the ESV1 and tLESV forms data to explain the function of both proteins. However, the analysis of the results revealed that LESV and ESV1 proteins bind strongly at the starch granule surface. Furthermore, not all of both proteins were released after their incubation with starches after washing the granules with 2% [w/v] SDS indicates to their binding to the deeper layers of the granule surface. Supporting of this finding comes after the binding of both proteins to starches after removing the free glucans chains from the surface by the action of ISA and BAM. Although both proteins are capable of binding to the starch structure, only LESV showed binding to amylose, while in ESV1, binding was not observed. The alteration of glucan structures at the starch granule surface is essential for the incorporation of phosphate into starch granule while the phosphorylation of starch by GWD and PWD increased after removing the free glucan chains by ISA. Furthermore, PWD showed the possibility of starch phosphorylation without prephosphorylation by GWD. Biochemical studies on protein-glucan interactions between LESV or ESV1 with different types of starch showed a potentially important mechanism of regulating and adjusting the phosphorylation process while the binding of LESV and ESV1 leads to altering the glucan structures of starches, hence, render the effect of the action of dikinases enzymes (GWD and PWD) more able to control the rate of starch degradation. Despite the presence of ESV1 which revealed an antagonistic effect on the PWD action as the PWD action was decreased without prephosphorylation by GWD and increased after prephosphorylation by GWD (Chapter 4), PWD showed a significant reduction in its action with or without prephosphorylation by GWD in the presence of ESV1 whether separately or together with LESV (Chapter 5). However, the presence of LESV and ESV1 together revealed the same effect compared to the effect of each one alone on the phosphorylation process, therefore it is difficult to distinguish the specific function between them. However, non-interactions were detected between LESV and ESV1 or between each of them with GWD and PWD or between GWD and PWD indicating the independent work for these proteins. It was also observed that the alteration of the starch structure by LESV and ESV1 plays a role in adjusting starch degradation rates not only by affecting the dikinases but also by affecting some of the hydrolysing enzymes since it was found that the presence of LESV and ESV1leads to the reduction of the action of BAM, but does not abolish it.…
• Ziel dieser Arbeit war es, den Mechanismus der Protein-Glucan-Wechselwirkungen bei der Regulation und Kontrolle des Stärkeabbaus zu verstehen. Der Stärkeabbau beginnt mit dem Phosphorylierungsprozess, der von den beiden Dikinasen, der a-Glucan, Wasserdikinase (GWD) und der Phosphoglucanwasserdikinase (PWD) durchgeführt wird. Kürzlich wurden einige Proteine gefunden, die mit dem Stärkegranulum assoziiert sind. Zwei dieser Proteine heißen Early Starvation 1 (ESV1) und das Homolog Like-Early Starvation (LESV), Es wurde vorgeschlagen, dass beide an der Kontrolle des Stärkeabbaus beteiligt sind, aber ihre Funktion ist bisher nicht bekannt. Um zu verstehen, wie ESV1- und LESV-Glucan-Wechselwirkungen reguliert werden und den Stärkeabbau beeinflussen, wurde der Einfluss der beiden Proteine auf die Phosphorylierungsenzyme GWD und PWD, sowie die Hydrolasen isoamylase, betaamylase, und alpha-amylase ntersucht. Dabei ergab die Analyse, dass LESV und ESV1 nicht nur stark an der Oberfläche, sondern auch in den tieferen Schichten derZiel dieser Arbeit war es, den Mechanismus der Protein-Glucan-Wechselwirkungen bei der Regulation und Kontrolle des Stärkeabbaus zu verstehen. Der Stärkeabbau beginnt mit dem Phosphorylierungsprozess, der von den beiden Dikinasen, der a-Glucan, Wasserdikinase (GWD) und der Phosphoglucanwasserdikinase (PWD) durchgeführt wird. Kürzlich wurden einige Proteine gefunden, die mit dem Stärkegranulum assoziiert sind. Zwei dieser Proteine heißen Early Starvation 1 (ESV1) und das Homolog Like-Early Starvation (LESV), Es wurde vorgeschlagen, dass beide an der Kontrolle des Stärkeabbaus beteiligt sind, aber ihre Funktion ist bisher nicht bekannt. Um zu verstehen, wie ESV1- und LESV-Glucan-Wechselwirkungen reguliert werden und den Stärkeabbau beeinflussen, wurde der Einfluss der beiden Proteine auf die Phosphorylierungsenzyme GWD und PWD, sowie die Hydrolasen isoamylase, betaamylase, und alpha-amylase ntersucht. Dabei ergab die Analyse, dass LESV und ESV1 nicht nur stark an der Oberfläche, sondern auch in den tieferen Schichten der Stärkegranula binden. Obwohl beide Proteine in der Lage sind, an die Stärkestruktur zu binden, zeigte nur LESV eine Bindung an Amylose, während für ESV1 keine Bindung beobachtet werden konnte. Die Veränderung der Glucanstrukturen an der Oberfläche der Stärkekörner ist für den Einbau von Phosphat wesentlich, so nahm beispielsweise die Phosphorylierung der Stärke durch GWD und PWD nach Entfernung der freien Glucanketten mittels ISA zu. Darüber hinaus konnte ebenso gezeigt werden, dass PWD auch ohne eine Präphosphorylierung durch GWD die Glucosyleinheiten innerhalb der Stärke phosphorylieren kann. Die Bindung von LESV und ESV1 führt zu einer Veränderung der Glucanstrukturen von Stärken, wodurch die Aktivität der Dikinasen (GWD und PWD) und somit die Geschwindigkeit des Stärkeabbaus wahrscheinlich besser gesteuert werden kann. Es wurden keine Wechselwirkungen zwischen LESV und ESV1 oder zwischen jedem von ihnen mit GWD und PWD oder zwischen GWD und PWD festgestellt, was auf die unabhängige Arbeit von diesen Proteinen hinweist. Es wurde auch beobachtet, dass die Modifikation der Stärkestruktur durch LESV und ESV1 eine Rolle bei der Anpassung der Stärkeabbauraten spielt, nicht nur durch Beeinflussung der Dikinasen, sondern auch durch die Beeinflussung einiger hydrolysierender Enzyme wie BAM. Den so zeigte die Amylase eine eindeutige Reduktion ihrer katalytischen Wirkung in Präsenz von LESV und ESV1. Daraus resumierend kann davon ausgegangen werden, dass die beiden Proteine ESV1 und LESV für die Feinregulation des Stärkeabbaus von höchster Relevanz sind.…