FU Berlin Digitale Dissertation
Ingo Przylas : Crystal structure analysis of Amylomaltase from Thermus aquaticus Die erste dreidimensionale Struktur einer Glycosyltransferase,die die Bildung großer zyklischer Amylose katalysiert Kristallstrukturanalyse der Amylomaltase aus Thermus aquaticus
|Abstract| |Table of Contents| |More Information|

Abstract 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.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title 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 Literaturverzeichnis

More Information:
Online available:	http://www.diss.fu-berlin.de/2001/15/indexe.html
Language of PhDThesis:	german
Keywords:	(beta, alpha)8-barrel; glucanotransferase; disproportionating enzyme; alpha-amylase-family
DNB-Sachgruppe:	30 Chemie
Date of disputation:	15-Dec-2000
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Prof. Dr. Wolfram Saenger
Second Referee:	Prof. Dr. Wolfgang Höhne
Contact (Author):	iprzylas@yahoo.com
Contact (Advisor):	saenger@chemie.fu-berlin.de
Date created:	06-Feb-2001
Date available:	13-Feb-2001

 

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