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- Furlanetto, Valentina
(författare)
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Symbiotic and pathogenic factors in plant-microbe interaction: Structural basis of C-glycoside metabolism and lipoprotein transport in bacteria
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The communication between plants and bacteria involves a complex chemical signaling network that mediates responses to various biotic and abiotic stresses, as well as establishing symbiotic relationships between different organisms.The first part of the thesis focuses on a mechanism for symbiotic communication between plants and bacteria and more specifically on how C-glycosylated aromatic polyketide compounds produced by plants can be used as a mechanism for plants to communicate with beneficial bacteria. In their glycosylated form, these compounds are substrates for symbiotic bacteria, which in return deglycosylate them and release the sugar-free part, the active aglycone. The aglycone can then mediate several functions beneficial to the plant, for example facilitating nitrogen fixation or acting as an antibacterial agent against plant pathogens.Results from studies covered in the thesis show that the soil bacteria Deinococcus aerius, Streptomyces canus and Microbacterium testaceum produce enzymes that can cleave the carbon-carbon bond between the sugar and the aglycone in C-glycosyl compounds. Deglycosylation first requires oxidation of the sugar by an oxidoreductase, after which the carbon-carbon bond can be cleaved by a C-glycosyl deglycosidase (CGD). Biochemical and structural characterization as well as results from phylogenetic analyzes of the amino-acid sequences of CGD enzymes provided new knowledge about these relatively unexplored enzymatic processes, as well as increased insight into how C-glycosylated aromatic polyketides participate in the interaction between plant and bacteria.The second part of the thesis explores pathogenic interactions between plants and bacteria. The virulence of pathogenic bacteria is dependent on lipoproteins that are attached to the bacteria's outer membrane and that have a decisive role for the bacteria's survival and pathogenicity. The localization of lipoproteins takes place through a process abbreviated Lol. The Lol system of the notorious plant pathogen Xanthomonas campestris was studied to better understand the underlying molecular mechanisms of the localization system, which could eventually open new ways to combat the bacterium. Biochemical, structural, and phylogenetic techniques were used also in this project.Taken together, the results contributed several new discoveries. For the first time, a physical complex between the two proteins responsible for transporting lipoproteins could be determined and their mutual interactions studied. Furthermore, sequence analyses challenge the generally accepted model of how lipoproteins are released from the bacterial inner membrane before being transported to the outer membrane. According to the standard model based on Escherichia coli, lipoproteins are extracted from the inner membrane by a membrane protein that belongs to the ABC-transporter family and whose structure forms an asymmetric heterodimer (LolCDE). However, our bioinformatic analysis show that most of these ABC transporters, including X. campestris, are likely to be homodimers and that Escherichia coli is the exception rather than the rule. The difference between an asymmetric and symmetric ABC transporter also has implications for several hypotheses about how these proteins function. Heterologous production of the X. campestris ABC transporter confirmed that the protein is a homodimer.
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| 2. |
- Hassan, Noor, 1977-
(författare)
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Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.
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