Abstract
A noncanonical route for p-aminobenzoate (pABA) biosynthesis in select bacteria utilizes a novel self-sacrificing heme oxygenase-like domain-containing oxidase/oxygenase (HDO) superfamily member. The recently characterized self-sacrificing pABA synthase from Chlamydia trachomatis ("CADD") requires manganese and likely employs a heterobimetallic Mn/Fe cofactor. A conserved active site tyrosine residue is cleaved from the protein backbone to serve as the substrate for pABA synthesis and a lysine residue is the amino group donor. Here, we investigated the orthologous pABA synthase from the ammonia-oxidizing bacterium, Nitrosomonas europaea, which we refer to as NePabS. Consistent with the previously studied C. trachomatis enzyme, purified NePabS produces pABA in vitro in a reaction that only requires a metal cofactor, molecular oxygen, and a reducing agent, but no other substrates. Interestingly, maximal activity was observed with the addition of only iron as opposed to manganese and iron; thus, NePabS utilizes the more traditional Fe/Fe cofactor employed by most characterized HDO superfamily members. The self-sacrificing residues were confirmed to be Tyr25 and Lys159, which are the corresponding self-sacrificing residues in the CADD reaction. Strikingly, we could switch the metal dependence (Fe/Fe to Mn/Fe) and significantly improve the activity (~ twofold) of NePabS by substituting two phenylalanine residues with tyrosine residues (F148Y/F177Y), thus rendering the enzyme more similar to CADD. These results demonstrate that these two aromatic residues play an essential role in dictating metal specificity and potentially the proposed radical translocation process that facilitates the tyrosine cleavage reaction for pABA synthesis.