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.
CADD (chlamydia protein associating with death domains) is a p-aminobenzoate synthase involved in a non-canonical route for tetrahydrofolate biosynthesis in the intracellular bacterial pathogen, Chlamydia trachomatis. The previously solved crystal structure revealed a seven-helix bundle architecture similar to heme oxygenase with a diiron active site, making CADD a founding member of the emerging HDO (heme-oxygenase-like diiron oxidase) superfamily. The CADD-dependent route for pAB biosynthesis was shown to use L-tyrosine as a precursor, however, in vitro reactions were not stimulated by the addition of free L-tyrosine or other tyrosine-derived metabolites, leading to the proposal that the enzyme uses an internal active site tyrosine residue as a precursor to pAB. Here, we perform further biochemical characterization of CADD to clarify the details of the unique self-sacrificing reaction. Surprisingly, the pAB synthase reaction was shown to be dependent on manganese as opposed to iron and the data are most consistent with an active dimanganese cofactor analogous to class Ib and class Id ribonucleotide reductases. Experiments with 18O2 demonstrated the incorporation of two oxygen atoms from molecular oxygen into the pAB product, supporting the proposed mechanism requiring two monooxygenase reactions. Mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, a modification that was increased in reactions that produced pAB in vitro. Additionally, Lys152 was found to be deaminated and oxidized to aminoadipic acid. Taken together, our results support the conclusion that CADD is a manganese-dependent oxygenase that uses Tyr27 and possibly Lys152 as sacrificial substrates for pAB biosynthesis.
Methanogenic archaea are strict anaerobes that produce methane as a byproduct of their essential energy metabolism known as methanogenesis. Tetrahydromethanopterin (H4MPT), a specialized cofactor similar to tetrahydrofolate (H4F), is a C1carrier required for the methanogenesis pathway. The biosynthesis of H4MPT utilizes a similar series of reactions used in H4F biosynthesis, but they are catalyzed by nonorthologous enzymes. Here, we report the recombinant expression, purification, and enzymatic properties of MptE (MJ1634), a novel 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) identified to catalyze the final step in the biosynthesis of the pterin portion in H4MPT in Methancaldococcus jannaschii by catalyzing the transfer of a pyrophosphate from ATP to the substrate dihydro‐6‐hydroxymethylpterin (H2HMP). This novel HPPK shows a distinctive sequence from the bacterial counterpart; instead, it belongs to the superfamily of thiamine pyrophosphokinase (TPK) catalytic domain. The gene encoding MptE from M. jannaschii was overexpressed in Escherichia coli, and the his‐tagged recombinant enzyme was purified by metal‐affinity chromatography. After purification, initial enzyme assays were performed and analyzed via HPLC, which revealed a similar catalytic efficiency to bacterial HPPKs. Site‐directed mutagenesis studies coupled with structural analyses revealed that Asp106 and Asp171 are essential for Mg2+ coordination, His129 and Phe169 play important roles in H2HMP binding, and Lys217 is important for transition state stabilization. These residues were selected because they are strictly conserved in the archaeal protein family COG1634 or thiamine pyrophosphokinase (TPK), whose active site resembles those in the COG1634 family. Currently, kinetic studies are underway for additional variants to further identify key residues of structural and catalytic importance.
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