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Presentación De Powerpoint Bacterial Chalcone Isomerases: Identification of a Thermostable Variant for Use in Flavonoids Biotechnology Elena Puerta-Fernández and Juan M. González. Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAs. CSIC). Funding: Thermostable Isomerase Processes for Biotechnology (TIPs). ERA-IB-16-049 Abstract CHI as a Key Enzyme in Only described in Plants Chalcone isomerases (CHI) are key enzymes for the synthesis of flavonoids, colorful Flavonoids Biosynthesis until 2004… molecules naturally present in plants with rising interest in Pharmaceutical and Food industries. Currently, flavonoids are either directly extracted from plants or Identified in prokaryotic Isolated from chemically synthesized. However, the chemical synthesis of some flavonoids is difficult and forbid the definition of “natural” in food industry, therefore increasing genomes Eubacterium ramulus their price. Developing a white biotechnological process for flavonoid synthesis will contribute to its sustainability, but requires finding robust and thermostable enzymes that would ensure its competitiveness. CHI CHIs catalyze the conversion between isomeric forms of chalcones, which are precursors of interesting molecules such as isoflavonoids, flavones, anthocyanins and tannins. CHIs were thought to be unique to plants until 2004 when they were also described in a few bacterial genomes. These previous works defined two types of bacterial chalcone isomerases: one with similarity to plant CHIs, identified mainly in Proteobacteria, and a second one, evolutionarily unique, identified in the Firmicutes Eubacterium ramulus (Era). However, only the bacterial CHI identified in Eubacterium was assayed for its catalytic activity. We have identified, through Bioinformatic searches, several representatives of both types of bacterial chalcone isomerases. Representatives of the first type, that we will name Shewanella-type, were identified mainly among Proteobacteria and Flavobacteria, with several moderate thermophilic representatives. The second type of bacterial chalcone isomerases, Eubacterium- Background type, were scarcely represented in bacterial genomes, and were almost exclusively restricted to Firmicutes. Both types of chalcone isomerases were purified after heterologous expression and assayed against two different substrates. The Shewanella-type chalcone isomerases showed only residual activity, both for Gensheimer, et al. Chalcone isomerase family Herles, et al. First bacterial chalcone isomerase mesophilic and thermophilic representatives. Eubacterium-type chalcone isomerases and fold: No longer unique to plants. isolated from Eubacterium ramulus. showed catalytic activity against both substrates, being bona fide chalcone Protein science (2004). 13:540-544 Arch Microbiol (2004) 184: 428-434 isomerases. In the Eubacterium group we identified one representative in a moderate Thomsen, et al. Structure and catalytic mechanism thermophile. The thermophilic CHI showed higher thermostability than the Era of the evolutionarily unique bacterial chalcone enzyme, with optimal catalytic rates at 70°C. The characteristics of this bacterial CHI isomerase. Acta Cryst (2015). D71, 907-917 makes it a good candidate for the development of a thermostable biotechnological process for flavonoid synthesis, opening a window of opportunity to design white Adapted from: Winkel-Shirley, B. Flavonoid Biosynthesis. A colorful model for Genetics, Biochemistry, Cell biology, and Biotechnology. biotechnological processes based on durable CHIs. Plant Physiol (2001) 126:485-493 Diversity Secondary Structure Model Flavobacterias Bacterial Predicted structure Eukarya Archaea Bacteria type Burkholderiales - Aquabacterium Ralstonia Thiomonas Duganella Thermoanaerobaculum Alteromonadales Oceanospirillales Shewanella Marinobacter Endozoicomonas Desulfacinum Sequence alignment of some of the proteins identified in our bioinformatics search. The secondary structure model is drawn based on Gensheimer, et al. Ferrimonas Moritella Hahella Thermodesulforhabdus Highlighted in yellow or in red are conserved residues according to Gensheimer, et al. Highlighted in green are important catalytic residues defined from M. sativa protein structure (crystalized). Notice that the residues important for catalysis in the plant CHI are absent in the predicted bacterial CHIs. One mesophile representative, from Shewanella baltica CHI protein sequence was used as Shewanella batica (Sba), and one thermophile representative, from Desulfacinum hydrothermale (Dhy), were selected for further characterization. Shewanella-type CHI query to identify bacterial CHI proteins. Positive BLAST hits were found mainly among Proteobacteria and Flavobacteria, with some thermophilic representatives among Oceanospirillales. Enzymatic Activity Colorimetric Assays Protein Purification Substrate: Naringenin Chalcone Substrate: Butein CHI activity at 25°C CHI Activity at 50°C Shewanella CHI Activity at 25°C CHI Activity at 50°C kDa 1,8 1,8 1,4 1,2 1,6 1,6 1,15 1,3 1,4 1,4 75 1,1 1,2 1,2 1,2 Naringenin chalcone: Yellow color; A368 Butein: Yellow color; A 1,05 368 63 1 1 368 368 368 368 1,1 A A A CHI-MBP 0,8 A 0,8 1 48 0,6 0,6 1 CHI CHI 0,95 CHI 0,4 0,4 0,9 0,9 0,2 0,2 0 0 0,8 0,85 Sba Dhy 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 70 0 5 10 15 20 25 0 5 10 15 20 25 time (min) time (min) time (min) time (min) Control Sba Dhy Control Sba Dhy Control Sba Dhy Control Dhy Naringenin: clear; no A368 Butin: Clear; no A368 Catalytic activity of putative CHIs were determined by a colorimetric assay, using either Naringenin Chalcone or Butein as substrates. These chalcones display a yellow color, with absorbance at 368 nm, whereas the corresponding flavanones are colorless. Activity is measured as the decrease in absorbance at 368 nm. Proteins were purified after heterologous expression in E. coli, as fusion proteins with a Maltose Binding Domain (MBP). Assays were performed either at 25°C or 50°C. Only residual activity can be detected when butein is used as substrate, and they might be ancestors of plant proteins called CHIL (Chalcone-isomerases like), that show structural similarity with CHI but lack its catalytic activity. Diversity Secondary Structure Model Thermoanaerobacteriales Clostridiales Secondary structure2 Eukarya Secondary structure 2 Bacteria type - Archaea Halotalea Holophaga 2Thomsen, et al. Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase. Acta Cryst (2015). D71, 907-917. Eubacterium ramulus CHI protein sequence was used as Eubacterium-type CHI A sequence alignment of some of the identified proteins showed that important residues for catalysis (highlighted in green) and for dimerization (in blue) were conserved in query to identify bacterial CHI proteins. Positive BLAST hits were found almost the identified proteins, suggesting a CHI catalytic role for these proteins. exclusively in the Firmicutes family, with a moderate thermophile representative (Tepidanaerobacter acetatoxydans). A representative protein from each group was purified and assayed for its catalytic activity. Enzymatic Activity Substrate: Butein Substrate: Naringenin chalcone A B CHI Activity at Different Temperatures A B Thermal stability at 50°C C CHI Activity at 60°C CHI Activity at High Temperatures -1 2 CHI kobs (min ) t1/2 (min) 1,2 2,5 0,16 1,8 0,14 Eubacterium 1 1,6 Era 1.42 0.49 2 0,12 1,4 ) 1 0,8 - 1,2 0,1 Tace 1.10 0.63 1,5 0,6 368 1 (min 368 0,08 A 0,8 A 1 obs 0,06 0,4 k Hfo 0.26 2.61 0,6 0,04 Relative CHI activityCHI Relative 0,2 0,4 0,5 0,2 0,02 Hal 0.27 2.58 0 0 50 100 150 200 250 300 0 0 0 CHI 0 2 4 6 30 40 50 60 70 80 90 Time at 50°C (min) 0 2 4 6 8 10 12 Aba 0.12 5.82 time (min) time (min) Temperature (°C) Era Tace Hal Hfo Control Tace Era Era 70°C Era 80°C Era 90°C Era Tace Tace 70°C Tace 80°C Tace 90°C A. Catalytic rates at room temperature using Naringenin chalcone as substrate (40µM) and 0.07 µg of protein per assay (100µl). Era: A. Era and Tace CHI activity at high temperatures using Butein as substrate. As expected, the Tace CHI protein Eubacterium ramulus CHI protein; Tace: Tepidanaerobacter acetotoxydans; Hfo: Holophaga foetida; Hal: Halotalea alkalilenta and Aba: shows better activity than the Era protein at high temperatures. B. Tace and Era CHI proteins catalytic rates Actinobacteria bacterium. B. Thermal stability of different proteins at 50°C. Results showed better thermostability for the Tace CHI than (kobs) at different temperatures. The optimal temperature for the Era CHI is 40°C, whereas the Tace CHI the Era protein. C. CHI activity for Era and Tace proteins at 60°C. Both proteins are active, although the substrate Naringenin chalcone is shows optimal activity at 70°C. highly unstable at this temperature, indicating the necessity of using an alternative substrate for assays at high temperatures..
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