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

Abstract

Chlamydia protein associating with death domains (CADD), the founding member of a recently discovered class of nonheme dimetal enzymes termed hemeoxygenase-like dimetaloxidases (HDOs), plays an indispensable role in pathogen survival. CADD orchestrates the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate via the self-sacrificial excision of a proteinderived tyrosine (Tyr27) and several additional processing steps, the nature and timing of which have yet to be fully clarified. Nuclear magnetic resonance (NMR) and proteomics approaches reveal the source and probable timing of amine installation by a neighboring lysine (Lys152). Turnover studies using limiting O 2 have identified a para-aminobenzaldehyde (pABCHO) metabolic intermediate that is formed on the path to pABA formation. The use of pABCHO and other probe substrates shows that the heterobimetallic Fe/Mn form of the enzyme is capable of oxygen insertion to generate the pABA-carboxylate.

Structural Basis for Methine Excision by a Heme Oxygenase-like Enzyme

“…The regeneration of the ferrous state is characteristic of mononuclear non-heme Fe-dependent oxidases, , but unlike that of other characterized HDOs, in which the diferric cofactor that forms following turnover needs to be reduced by an auxiliary reducing system. For instance, SznF, UndA, and BesC all require a reducing agent to catalyze multiple turnovers, while CADD only performs a single turnover as a suicide enzyme. − Although the Fe cofactor in FlcD is labile and the protein is isolated in its apo form, Fe does not appear to be a cosubstrate, as higher levels do not enhance formation of intermediates or product, signifying another difference with the prototypical HDOs. Cumulatively, our spectroscopic and biochemical findings show that FlcD employs a mononuclear non-heme Fe cofactor that breaks the paradigm of a dinuclear active site for the HDO family.…”

“…Our results indicate that FlcD is a remarkable mononuclear iron enzyme in the heme oxygenase-like superfamily. The mononuclear iron cofactor is distinct from other characterized members of the superfamily, which currently include dinuclear Fe (SznF, UndA, BesC, and AetD), ,,,,,, dinuclear Fe/Mn or Mn/Mn (CADD), − and no metal cofactor (PqqC). , As in the case of FlcD, deviations in iron-binding motifs may be used to predict the cofactor nuclearity in heme oxygenase-like enzymes. The low frequency and high conservation of the mononuclear iron-binding motif of the FlcD subgroup (∼1% of all groups) suggest that mononuclear iron-dependent enzymes constitute a small fraction of the sequenced HDOs.…”

“… − C hlamydia protein a ssociating with d eath d omains (CADD) catalyzes the cleavage between the α- and β-carbon of its active site tyrosine and an amination to generate para -aminobenzoic acid (Figure S1C). − HDOs also introduce essential functionalities into the structures of therapeutically and ecologically important natural products. For example, SznF catalyzes consecutive N -hydroxylations of methylarginine in the biosynthesis of the anticancer agent streptozotocin (Figure S1B).…”

“…Structural analysis of these HDOs revealed a conserved motif that is responsible for binding the dinuclear metal cofactor. However, activity of HDOs is not solely iron-dependent, as CADD appears to employ a dinuclear Mn/Fe cofactor for catalysis. − …”