CT610: A Mn-Dependent Self-Sacrificing Oxygenase in p-Aminobenzoate Biosynthesis in Chlamydia trachomatis

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.