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

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.