Structural and biochemical studies on enzymes involved in natural product biosynthesis and cellular respiration

X-Ray Crystallography;Natural Products, Protein;Enzyme Structure;Drug Design;Membrane Protein;Protein Purification;Alginate Lyase;Plumbemycin;Rhizocticin;Antibiotic;Antifungal;aa3-Menaquinone Oxidase;Mining Microbial Genomics;Biofuels;Biodiesel;Enzyme Kinetics;Electron Transport Chain

Park, David S

关键词: X-Ray Crystallography;    Natural Products, Protein;    Enzyme Structure;    Drug Design;    Membrane Protein;    Protein Purification;    Alginate Lyase;    Plumbemycin;    Rhizocticin;    Antibiotic;    Antifungal;    aa3-Menaquinone Oxidase;    Mining Microbial Genomics;    Biofuels;    Biodiesel;    Enzyme Kinetics;    Electron Transport Chain;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/90469/PARK-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Natural products have recently become an interest in modern research. This is due to their synthesis mainly by living organisms and their natural biosynthesis and abundance. Many of these compounds have been found to have pharmaceutical and industrial applications, and have been used as the basis for derivatives that are already being used for large scale production. Despite initial availability, there now is an urgent need to isolate and produce new and more effective natural products. The good news is we are still continuing to discover new sources. Natural products will still be an important source for industrial and pharmaceutical development. One focus improving them is to understand the biosynthetic pathways of some of these natural products. Elucidating these biosynthetic pathways will allow us to be improve efficacy and allow for rational design of new and better compounds. The studies that were conducted in this dissertation focuses on structural studies of three enzymes involved with natural product synthesis.The most widely distributed component of brown algae is alginate, a cell wall polysaccharide composed of polymeric blocks of α-(1,4) O-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G). Biochemical characterization of the Saccharophagus degredans polysaccharide lyase-17 familty enzyme (Alg17c) demonstrates that the enzyme can depolymerize alginate di-, tri-, and tetra-saccharide into monosaccharides, providing the necessary precursor for ethanol fermentation. In chapter 1 we present several crystal structures of the PL-17 enzyme Alg7c from S. degredans, including the wild-type enzymes, respective mutants, and a co-crystal structure of one of the varients in complex with an alginate trisaccharide. Structure-guidedanalysis of several active site variants, allow for the identification of residues that are critical for substrate recognition and for the bases of the exolytic reaction mechanism of PL-17 enzymes.Plumbemycin is an antibacterial phosphonate peptide from Streptomyces plumbeus containing APPA (2-amino-5-phosphono-3-pentenoic acid), a compound that inhibits the threonine synthesis. The current pathway is unknown, but a proposed pathway has been formulated based on the pathway of a structurally similar APPA containing antifungal compound rhizocticin. There is a putative class II aldolase, PluG, in the biosynthetic pathway which is proposed to catalyze an aldol reaction between oxaloacetate and phosphonoacetaldehyde to form a reactive hydroxyl intermediate (2-keto-4-hydroxy-5-phosphonopentanoic acid), with a chance to form an irreversible, non-enzymatic dehydration reaction which forms an off pathway end product. In chapter 2, we obtained a high resolution structure of a PluG ortholog from Saccharothrix aerocolonigenes. The data from this structure shows an uncommon c-terminal domain, which we hypothesize to promote the formation of a complex with a subsequent enzyme from the plumbemycin biosynthetic pathway. We also propose that a hydrophobic tunnel forms between the two active sites of the complex and propose that this tunnel shuttles the reactive hydroxyl intermediate, thus preventing the formation of the non-enzymatic end product.Cellular respiration is a critical components of a cellular organism’s ability to adapt and survive in its environment, as well as produce potentially useful natural products. It is not coincidence that many researchers are focusing on elucidating pathways involved with this process, especially that of the electron transport chain. B. subtilis is also one of the most heavily studied and utilized organisms, with its genome completely sequenced and easily found in soil, gastrointestinal tracts of humans, and other readily accessible sources. However chemistry involved with the electron transport chain still has many questions that need to be answered. We carried out structural studies on the aa3-menaquinone oxidase, which has high sequence similarity with ubiquinone oxidase from E. coli. We believe structural information on the aa3 can elucidate the mystery of the quinone/menaquinone binding site, which is critical for the q cycle in the electron transport chain of bacteria.