Die erste dreidimensionale Struktur einer Glycosyltransferase,die die Bildung großer zyklischer Amylose katalysiert

Titelblatt Einleitung 1.1 Polysaccharid 1.2 Metabolismus der Stärke 1.3 Die Amylomaltase 1.4 Industrielle Anwendung der Stärke-produzierenden Enzyme und deren Produkte 1.5 Zielsetzung Material und Methoden 2.1 Materialien 2.2 Biochemische Methoden 2.3 Kristallographische Methoden Ergebnisse 3.1 Proteinchemische Untersuchungen 3.2 Strukturlösung Strukturanalyse 4.1 Tertiärstruktur und Hauptkettenverlauf 4.2 Amylomaltase in Komplex und Acarbose 4.3 Kristallkontakte 4.4 Thermostabilität und Temperaturfaktoren Diskussion und Schlussfolgerungen 5.1 Vergleich der nativen und Inhibitor-gebundenen Struktur 5.2 Sequenzalignment verschiedener Amylomaltasen und eines D-Enzyms 5.3 Vergleich der Struktur der Amylomaltase mit verwandten Strukturen aus der Alpha-Amylase-Familie 5.4 Diskussion der Inhibitor-gebundenen Struktur 5.5 Möglicher Bindungspfad längerer Substrate und Mechanismus der Ringbildung Zusammenfassung und Ausblick AnhangKohlenhydrate sind essentielle Stoffe aller Organismen und bilden die variabelste Klasse aller natürlich vorkommenden Moleküle. Polysaccharide wie Stärke sind wichtige Energie-speicher in Pflanzen, Bakterien und einfacheren Eukaryonten. Für Tiere ist Stärke ein Haupt-nahrungs-mittel, die in Form von Glykogen gespeichert wird. Die Bildung bzw. die Hydrolyse der glycosidischen Bindung ist daher für alle Organismen ein kritischer Schritt bei der Energie Aufnahme und Speicherung, wodurch sich die Vielzahl der Stärke-pro-zessieren- der Enzyme erklärt. Amylomaltasen als Mitglied der a-Amylase-Familie katalysiert die Trans-glykosi-lie-rung von a-1,4-Glucanen. Die Struktur der Amylomaltase aus Thermus aquaticus wurde röntgen-kristallo-graphisch mittels des multiplen Isomorphen-Ersatzes bis 2.0 Å in seiner nativen Form und bis 1.9 Å in Komplex mit Acar-bose, einem Maltotetraosederivat, untersucht. Die Amylomaltase enthält wie alle Mitglieder der a-Amylase-Familie ein (b, a)8-Faß, dessen regel-mäßige Abfolge der Sekundärstrukturelemente durch mehrere Einschübe unterbrochen wird. Große Insertionen befinden sich einen zwischen dem dritten und fünften Faßstrang und formen die Unterdomäne B1. Die Unterdomäne B2 wird durch die langen a-helikalen Unter-brechungen zwischen dem zweiten und dritten Faßstrang gebildet. In anderen bekannten Strukturen der a -Amylase-Familie sind lediglich Teile der Unter-domäne B2 vor-handen. Die Unterdomäne B1 wurde bisher in keinem anderen Enzym dieser Familie gefunden. Desweiteren hat die Amylomaltase keine C-terminale Domäne C, die in allen anderen Strukturen vorkommt und für deren katalytische Aktivität notwendig ist. Die homologe Anordung der katalytisch aktiven Seitenketten Aspartat-293, Glutamat-340 und Aspartat-395 der Amylomaltase im Vergleich zu bekannten Proteinen der a-Amylase-Familie weist auf einen ähnlichen Reaktionsmechanismus für die Trans-glyko-silierung hin. Eine besomdere Eigenschaft der Amylomaltase ist die Katalyse der Bildung von großen zyklischen Ringen. Diese Ringe haben im Falle der Amylomaltase aus Thermus aquaticus einen Polymerisationsgrad größer als 22 Einheiten. Die sehr gut charakteriserten Cyclodextrin Glucanotransferasen (CGTasen) katalysieren lediglich die Bildung Ringe zwischen 6 und 8 Glucosen. Aufbauend auf den zwei Bindungsstellen der Acarbose in der Inhibitor-gebundenen Struktur und dem Vergleich der molekularen Oberfläche mit verwandten Enzymen konnte eine Hypo-these für den Bindungsmodus von längeren Amyloseketten aufgestellt werden, so daß mögliche Kandidaten, die die Ringgröße und deren Ausbeute beeinflussen erhalten wurden.Carbohydrates are essential components of all living organisms and form the most abundant class of biological molecules. Polysaccharides such as starch are an important food reserve in plants and a major nutrient for animals. Whereas higher plants synthesize starch, bacteria, lower eukaryotes and animals accumulate glycogen. Due to the important biological role of these poly-saccharides for energy storage and uptake, selective hydrolysis and formation of glycosidic bonds are critical steps for all organisms. Thus, various enzymes have been identified to act on starch. Amylo-maltase catalyses the transglycosylation reaction of a-1,4-glucans and is a member of the a-amylase family of enzymes. The crystal structure of amylomaltase from Thermus aquaticus was determined by multiple iso-morphous replacement to 2.0 Å resolution and in complex with acarbose, a maltotetraose derivative, to 1.9 Å resolution. As a member of the a-amylase family the core structure of amylo- maltase consists of a (b, a)8 barrel. In amylomaltase, the eight-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the a-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the a-amylase family, subdomain B2 is unique to amylo-maltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the a-amylase family and essential for their catalytic activity, is missing in amylo-maltase. The catalytic side chains (two Asp and one Glu) of amylomaltase show a similar arrange-ment as in previously characterized members of the a-amylase family, indicating similar mechanisms of the glycosyl transfer reaction. A unique feature of amylomaltase is its ability to catalyse the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucano-transferases (CGTases), which synthesize cycloamylose with a ring size of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a size of 22 glucose residues and higher. In the inhibitor bound structure of amylomaltase, two binding sites for acarbose were located. The analysis of these binding sites, the molecular surface and a comparison to related amylomaltase sequences revealed a possible binding mode for large amylose substrates and suggested candidates for amino acids, Tyr-54 and amino acids within the 250s and 460s loop, which might be varied by muta- genesis in order to influence the cyclization yield and the product ring size

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these -glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in -glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4- -glucan 4- -glucosyltransferase from GH31. Distinct from 1,4- -glucan 6- -glucosyltransferases (EC 2.4.1.24) and 4- -glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from (134)- glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro--glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme

Reinforced HNA backbone hydration in the crystal structure of a decameric HNA/RNA hybrid

The crystal structure of a decameric HNA/RNA (HNA = 2`,3`-dideoxy-l`,5`-anhydro-D-arabinohexitol nucleic acid) hybrid with the RNA sequence 5`-GGCAUUACGG-3` is the first crystal structure of a hybrid duplex between a naturally occurring nucleic acid and a strand, which is fully modified to contain a six-membered ring instead of ribose. The presence of four duplex helices in the asymmetric unit allows for a detailed discussion of hydration, which revealed a tighter spinelike backbone hydration for the HNA than for the RNA-strands. The reinforced backbone hydration is suggested to contribute significantly to the exceptional stability of HNA-containing duplexes and might be one of the causes for the evolutionary preference for ribose-derived nucleic acids

Reinforced HNA backbone hydration in the crystal structure of a decameric HNA/RNA hybrid

The crystal structure of a decameric HNA/RNA (HNA = 2',3'-dideoxy-1',5'-anhydro-d-arabinohexitol nucleic acid) hybrid with the RNA sequence 5'-GGCAUUACGG-3' is the first crystal structure of a hybrid duplex between a naturally occurring nucleic acid and a strand, which is fully modified to contain a six-membered ring instead of ribose. The presence of four duplex helices in the asymmetric unit allows for a detailed discussion of hydration, which revealed a tighter spinelike backbone hydration for the HNA- than for the RNA-strands. The reinforced backbone hydration is suggested to contribute significantly to the exceptional stability of HNA-containing duplexes and might be one of the causes for the evolutionary preference for ribose-derived nucleic acids.status: publishe

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf