Folate is an essential cofactor required for several processes including DNA and amino acid biosynthesis. Folate molecules are made up of three parts: a pteridine ring, p-aminobenzoate (pABA), and a variable number of glutamate residues. Chlamydia trachomatis synthesizes folate de novo; however, several genes encoding enzymes required for the canonical folate biosynthesis pathway are missing, including pabA/B and pabC, which are normally required for pABA biosynthesis from chorismate. Previous studies have found that a single gene in C. trachomatis, CT610, functionally replaces the canonical pABA biosynthesis genes. Interestingly, CT610 does not use chorismate as a substrate. Instead, the CT610-route for pABA biosynthesis incorporates isotopically labeled tyrosine into the synthesized pABA molecule. However, in vitro experiments revealed that CT610 produces pABA without any added substrates (including tyrosine) in the presence of a reducing agent and molecular oxygen. CT610 shares low sequence similarity to non-heme diiron oxygenases and the previously solved crystal structure revealed a diiron active site. Taken together, CT610 is proposed to be a novel self-sacrificing enzyme that uses one of its active site tyrosine residues as a precursor to pABA in a reaction that requires O2 and a reduced metallocofactor. Here, we discuss our progress towards understanding CT610-catalyzed pABA synthesis. Upon investigation of the pABA production and oxygenase activities of several active site tyrosine to phenylalanine variants, we found that Y27 and/or Y43 are the most likely precursors to the resulting pABA molecule. Further, activity was nearly completely abolished with a K152R variant, suggesting that this conserved lysine may be the required amino group donor. We also developed an in vitro Fe(II) reconstitution procedure, where the reconstituted enzyme exhibited a drastic increase in oxygenase activity but, surprisingly, a significant decrease in pABA synthase activity. Interestingly, a significant increase in pABA synthase activity was observed when the enzyme was reconstituted with manganese as opposed to iron, suggesting that the diiron active site of this enzyme might not be directly involved in CT610-dependent production of pABA and instead Mn may be the actual cofactor. Finally, we show that two 18O atoms from molecular oxygen are incorporated into the pABA molecule when synthesized by Mn-reconstituted CT610, providing further evidence for the oxygenase activity of CT610 and supporting our proposed mechanism that involves two monooxygenase reactions. / Master of Science in Life Sciences / Folate is an essential molecule that is required for all cells to survive. Folate is usually made in the cell with the help from proteins known as enzymes. Enzymes help biochemical reactions happen by speeding up the rate of their specific chemical reaction. In order for this to occur, an enzyme binds to a very specific molecule, called a substrate, and facilitates the reaction transforming the substrate into a new product while not altering the enzyme in the process, allowing for the protein to continuously facilitate this reaction. Chlamydia trachomatis is the strain of bacteria that causes one of the most common sexually transmitted infections in the US, Chlamydia. These bacteria make folate themselves but have been shown to make this molecule in a very different way from an average folate-synthesizing organism. One enzyme in C. trachomatis known as CT610 has been shown to participate in this unusual route to produce folate. Interestingly, CT610 is thought to remove part of itself to donate to the molecule it produces, effectively killing the enzyme after only one reaction. In this study we show that CT610 performs very unique chemistry to ultimately facilitate the production of folate to allow C. trachomatis to survive. This knowledge could be used in the future for the design of antibiotics specifically targeting C. trachomatis and thus treating the infections caused by this organism.

Tetrahydrofolate (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. / Master of Science in Life Sciences / Folate 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.