The Chlamydia trachomatis p‐aminobenzoate synthase CADD is a manganese‐dependent oxygenase that uses its own amino acid residues as substrates

CADD (chlamydia protein associating with death domains) is a p‐aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope‐labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry‐based proteomic analyses of CADD‐derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor. CADD is a p‐aminobenzoate (pAB) synthase involved in an unusual route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Here, we show that the enzyme is manganese‐dependent and likely employs a heterodinuclear Mn/Fe cofactor. Further, the substrate is demonstrated to be the enzyme itself, where the aromatic portion of Tyr27 and the amino group of Lys152 are sacrificed for pAB synthesis.