Biochemical Characterization of Self-Sacrificing P-Aminobenzoate Synthases from Chlamydia Trachomatis and Nitrosomonas Europaea

dc.contributor.authorStone, Spenseren
dc.contributor.committeechairAllen, Kylie Dawnen
dc.contributor.committeememberKennelly, Peter J.en
dc.contributor.committeememberLemkul, Justin Alanen
dc.description.abstractTetrahydrofolate (THF) is an essential cofactor for one-carbon transfer reactions in various biochemical pathways including DNA and amino acid biosynthesis. This cofactor is made up of three distinct moieties: a pteridine ring, p-aminobenzoate (pABA), and glutamate residues. Most bacteria and plants can synthesize folate de novo, unlike animals that obtain folate from their diet. An established pathway for THF biosynthesis exists in most bacteria, but there is evidence of some organisms such as Chlamydia trachomatis and Nitrosomonas europaea which do not contain the canonical THF biosynthesis genes, despite still being able to synthesize THF de novo. Previous studies have shown that these organisms do not contain the pabABC genes, normally required to synthesize the pABA portion of THF, and can circumvent their presence with just a single gene: ct610 and ne1434 from C. trachomatis and N. europaea, respectively. Interestingly, these novel enzymes for pABA synthesis do not use the canonical substrates, chorismate or other shikimate pathway intermediates. The gene product of ct610 was named Chlamydia Protein Associating with Death Domains (CADD) due to its established role in host mediated apoptosis, while the crystal structure showed an architecture similar to know diiron oxygenases. However, we provide evidence of a moonlighting function in pABA synthesis. Isotopic labeling experiments to understand what substrate might be used by CADD found that isotopically labeled tyrosine was incorporated into the final pABA product. Compellingly, CADD was able to produce pABA in the presence of molecular oxygen and a reducing agent alone without the addition of any exogenous substrate, implicating this unusual enzyme as a self-sacrificing pABA synthase from C. trachomatis. Here, we provide strong evidence for Tyr27 being a sacrificial residue that is cleaved from the protein backbone to serve as the pABA scaffold. Furthermore, we also provide evidence that K152 is an internal amino donor for this pABA synthase reaction performed by CADD. In the case of NE1434, we have conducted initial experiments such as site-directed mutagenesis and our findings suggest that these self-sacrificing residues are conserved between two distantly related organisms. Finally, the pABA synthase activity is reliant on an oxygenated dimetal cofactor and despite the crystal structure of CADD depicting a diiron active site, we have demonstrated that CADD's pABA synthase activity is dependent on a heterodinuclear Mn/Fe cofactor. Conversely, NE1434 demonstrates no preference for manganese and likely employs a more traditional Fe/Fe cofactor for catalysis. Our results implicate the CADD and NE1434 as self-sacrificing pABA synthases that have diverging metal requirements for catalysis.en
dc.description.abstractgeneralFolate is a molecule used by all organisms that is necessary for survival. Many kinds of bacteria are able to make this molecule with proteins called enzymes, which help by quickening the rate of a reaction. Enzymes are catalysts that usually work by binding a molecule, called a substrate, and will act on this substrate to generate a product; the enzyme remains unchanged in this process, which allows it to facilitate many more of these reactions. Chlamydia trachomatis, which is a leading cause of sexually transmitted infections (STIs) in the United States, and Nitrosomonas europaea, an environmental bacterium, are able to use enzymes to make their own folate, but not in the way that many other bacteria do. These organisms contain enzymes that use a part of their own structure as a substrate, making them "sacrificial lambs". Our study provides evidence of how these organisms carry out an abnormal chemical reaction to make folate which can help scientists target this pathway for the development of antibiotics.en
dc.description.degreeMaster of Science in Life Sciencesen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.subjectChlamydia trachomatisen
dc.subjectNitrosomonas europaeaen
dc.subjectself-sacrificing enzymeen
dc.subjectfolate biosynthesisen
dc.titleBiochemical Characterization of Self-Sacrificing P-Aminobenzoate Synthases from Chlamydia Trachomatis and Nitrosomonas Europaeaen
dc.typeThesisen Polytechnic Institute and State Universityen of Science in Life Sciencesen


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