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Title:Homeostasis of primary and secondary metabolites
Author(s):Olivares, Philip
Director of Research:Nair, Satish K.
Doctoral Committee Chair(s):Nair, Satish K.
Doctoral Committee Member(s):Metcalf, William W.; Procko, Erik; Zhang, Kai
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):X-ray
crystallography
antifreeze
protein
fosfomycin
natural products
metabolism
gamma-butyrolactones
Abstract:The ability for an organism to maintain homeostasis is of the utmost importance from the smallest single-celled lifeform, to the largest blue whale. While the needs of these organisms may vary widely based on their environments, analogous processes are at play. In this thesis, I will describe my doctoral work towards structurally and functionally understanding how a few of these systems, from single celled prokaryotes like streptomycetes and pseudomonads to an Antarctic fish, allow these organisms to survive and thrive in their respectively harsh environments. Starting on the smallest size scale and the uptake of nutrients for use in central metabolism by a single-celled organism from its environment, I investigate a solute binding protein called HtxB from Pseudomonas stutzeri WM88. This protein has been show to be involved in the uptake of reduced phosphorus compounds such as phosphite and hypophosphite when the more widely available phosphate is not available for utilization as a phosphorus source. Phosphorus, being needed for the synthesis of many biomolecules such as phospholipids, DNA, RNA, and ATP, can easily be the mass limiting element halting further cell division. In this research, I show high resolution crystal structures of HtxB bound to hypophosphite (1.95 Å), phosphite (1.03 Å), and methylphosphonate (1.14 Å), while also interrogating their binding through microscale thermophoresis and surface plasmon resonance. Moving on to the larger scale of intra-bacterial communication through signaling molecules, I investigate the crystal structures of ScbR2, a pseudo-gamma butyrolactone receptor, and AvaR1, the first known butenolide receptor. Both of these receptors are members of a larger class of homologous proteins found throughout streptomycetes involved in the regulation of secondary metabolism. Of these homologous transcriptional repressors, three main classes are known, while all respond to membrane diffusible secondary metabolites. The largest and most well studied class are the gamma-butyrolactone (GBL) receptors, named after the small molecule signals of which they bind and respond to. To the best of my knowledge, all characterized members of this class repress secondary metabolism through at least one biosynthetic gene cluster, while signal is not present. When the threshold concentration of the cognate ligand is reached, the repressor is released from DNA allowing downstream genes to be transcribed. Once transcription is no longer repressed, the organism can produce what are usually antibiotic compounds. The newest class of these repressors are the butenolide receptors, also named after the signaling molecules they bind and respond to, which while similar, have a difference cyclized head group. While only one butenolide receptor currently has an identified cognate ligand, it has generated significant interest as it involved in regulating avermectin production in Streptomyces avermitilis. A semi-synthetic analogue of avermectin, ivermectin shared the 2015 Nobel Prize for Physiology or Medicine. I describe the crystal structures of AvaR1 alone, and bound to either avenolide or a small double stranded DNA segment. Through various bioinformatic analyses, I have also been able to identify 63 sets of putative butenolide receptor and butenolide biosynthetic genes in various streptomycetes seen. Then I applied this information to the crystal structures obtained indicating a conserved butenolide ring and a more variable tail, mirroring the variability seen in analagous butanolide ligands. The third class of homologous repressors, the pseudo gamma-butyrolactone receptors, named not after the ligands in which they bind, but from the similarity in primary amino acid sequence to that of the first discovered bona-fide GBL receptors. Unlike the last two classes, the pseudo receptors are known to bind two or more, often structurally diverse secondary metabolites. This allows the host to integrate a variety of signals to result in the same downstream effect. Interestingly, these metabolites may be produced by their own colony, or neighboring colonies of another species. These receptors continue to differ from the first two classes such that they usually act to divert biosynthetic flux from the production of one secondary metabolite to another. Here I show the first crystal structure of a pseudo receptor, ScbR2. Unfortunately it is not bound to a ligand, however the lack of seven residues in the structure indicate the possibility of a mobile motif which presumably becomes structured upon ligand binding. Moving from the regulation of antibiotic biosynthesis, I discuss my efforts into understanding the function of Psf3, a stereospecific reductase that carries out the penultimate step of fosfomycin biosynthesis in Pseudomonas syringae. The crystal structure of Psf3 bound to its cofactor and substrate, along with mutational analysis confirm the function of the enzyme’s role in producing this medically relevant antibiotic. Lastly, I discuss the structure and function of an antifreeze-potentiating protein (AFPP) found in Pagothenia borchgrevinki, an Antarctic notothenioid fish endemic to the seas surrounding Antarctica, which often are close to -2° C. Clearly, for an organism comprised of a large amount of liquid water, some mechanism is needed to avoid ice formation inside its body to survive. While colligative properties are commonly known to suppress the freezing point of water, such high salt concentrations may hinder biological systems. Utilizing AFPPs the organisms can depress the freezing point of water 300 to 500 times more than calculated through the AFPPs colligative properties alone. AFPPs function by physically binding and inhibiting the growth of nucleating ice crystals. Such mechanisms are found in numerous organisms, from fish to insects, and have evolved independently on at least five separate occasions. Currently, four types of AFPPs are known in literature and classified by their structural folds. The AFPP reported here, is the first member of a fifth type, having a structure reminiscent of a C1q domain. With the combined structural information I have obtained throughout my doctoral research I have increase the knowledge of how organisms, from different environments and kingdoms of life maintain homeostasis. Whether these processes aid in obtaining needed nutrients from their environment, fending off unfriendly neighbors, or simply not being frozen solid, understanding these mechanisms may help future generations to more readily discover, or elicit the production of secondary metabolites in vitro and in vivo, and possibly even to find better food additives or create more frost resistant crops.
Issue Date:2019-03-13
Type:Text
URI:http://hdl.handle.net/2142/105142
Rights Information:Copyright 2019 Philip Olivares
Date Available in IDEALS:2019-08-23
Date Deposited:2019-05


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