Biochemische Charakterisierung von Ectoin-Biosynthese-Enzymen

Die Akkumulation von kompatiblen Soluten ist eine unter Mikroorganismen weit verbreitete Strategie zum Schutz vor dem Einfluss von hyperosmotischen Bedingungen in der Umgebung. Ectoin und sein Derivat 5-Hydroxyectoin sind prominente Mitglieder dieser Stressprotektiva. Aufgrund ihrer Eigenschaften we...

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Bibliographic Details
Main Author: Richter, Alexandra
Contributors: Bremer, Erhard (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2019
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Table of Contents: Among microorganisms, the accumulation of compatible solute is a widely used strategy to adapt to hyperosmotic conditions in the environment. Ectoine and 5-hydroxyectoine are prominent members of these stress protectants among bacteria. Due to their attributes they are described as „chemical chaperones“ and used for example in the pharmaceutical industry. The ectoine biosynthesis is performed by three enzymes. By a fourth enzyme reaction 5-hydroxyectoine can be produced. This work describes the biochemical and structural analysis of the L-2,4-diaminobutyrate transaminase EctB and the L-2,4-diaminobutyrate acetyltransferase EctA, which catalyze the first and the second reaction of the ectoine biosynthesis pathway. The conversion of L-aspartate-beta-semialdehyde and L-glutamate into diaminobutyrate (DAB) and 2-oxoglutarate is catalyzed by EctB and the following acetyl transfer from acetyl-CoA onto DAB, with N-gamma-acetyl-diaminobutyrate (N-gamma-ADABA) as product, is mediated by EctA. Therefore, the protein homologues of the thermotolerant bacterium Paenibacillus lautus were used. In this organism the ectoine biosynthesis genes are organized in an unusual combination with genes encoding an ABC-transport system, which was identified as an ectoine- / 5-hydroxyectoine specific Ehu-type transport system. This composition enables the resource-saving regulation of the ectoine level in the cytoplasm. The transaminase (Pl)EctB shows a high tolerance for fluctuations in pH, temperature and salt concentration. The PLP-dependent enzyme reaction, catalyzed by the tetrameric EctB protein, is reversible and the reverse reaction of this enzyme is very similar to the gamma-aminobutyrate transaminase (GABA-TA) reaction. An in silico modelling study revealed also a high structural relationship between these two enzymes. This was confirmed by the ability of (Pl)EctB to catalyze also the GABA-TA reaction. The acetyl-transferase (Pl)EctA catalyzes the highly regioselective formation of N-gamma-ADABA, whereas the isomer N-aplha-ADABA, which is an intermediate of the ectoine catabolism, is not formed. The five high-resolution crystal structures of (Pl)EctA were the basis for the analysis of the reactive site of the enzyme. By an additional site-directed mutagenesis study the amino acids involved in DAB-binding were identified. Both, EctB type and EctA type proteins, are evolutionarily conserved; therefore, the knowledge gained in this work is suitable as „blueprint“ for other members of these enzyme groups. Several organisms possess ectC* genes, existing independently from the classical ect gene cluster. The encoded EctC*-type proteins exhibit a high structural similarity to EctC type proteins, which catalyze the iron dependent reaction of N-gamma-ADABA producing ectoine. However the catalytic activity of these EctC*-type proteins is rudimentary, indicating that this enzyme reaction is not the main function of these enzymes. In summary, besides a first analysis of the EctC* type proteins, this work provides detailed information about the biochemistry and structure of the ectoine biosynthetic enzymes EctA and EctB. It therefore completes the structural and biochemical characterization of the entire ectoine / 5-hydroxyectoine biosynthetic pathway.