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

Abstract

Edited by Peter BrzezinskiCADD (chlamydia protein associating with death domains) is a paminobenzoate (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.

Excision of a Protein-Derived Amine for p-Aminobenzoate Assembly by the Self-Sacrificial Heterobimetallic Protein CADD

“…We next sought to further investigate the fate of Lys152 by using proteomics approaches. Another study reported the deamination and oxidation of this residue to aminoadipic acid, but results could not be specifically linked to catalysis due to the use of a low-activity preparation of the enzyme, sole-reconstitution with Mn, and additional modifications of Lys that can result from metal-catalyzed generation − of reactive oxygen species. This region eluded detection in our previous proteomics study because complete trypsin digestion of CADD is expected to result in a dipeptide (EK 152 ) with an m / z value of 276, which is beyond the MS1 scan range ( m / z = 375–1600) of the liquid chromatography–mass spectrometry/MS (LC-MS/MS) method that was used.…”

“…Unlike the HDOs that have been spectroscopically, ,,− , or in the case of SznF and AetD, structurally verified to house a diiron cofactor, a recent reexamination of CADD metal-dependence has shown that the enzyme requires both Fe and Mn for efficient p ABA production . A later study by Wooldridge and colleagues confirmed the essential role played by Mn for CADD activity . However, the most surprising facet of the p ABA assembly is that an exogenous substrate is not required.…”

“…23 A later study by Wooldridge and colleagues confirmed the essential role played by Mn for CADD activity. 24 However, the most surprising facet of the pABA assembly is that an exogenous substrate is not required. Isotopic-tracking and proteomics studies have shown that pABA results from the cleavage of Tyr27 that is located ∼14 Å from the dimetal site, and we proposed a complex sequence for pABA synthesis that includes the Cα-Cβ scission of Tyr27 via long-range radical transfer, cryptic amination from a source that has yet to be unambiguously identified, and more canonical oxygen insertion steps.…”