A Peroxodiiron(III/III) Intermediate Mediating Both N-Hydroxylation Steps in Biosynthesis of the N-Nitrosourea Pharmacophore of Streptozotocin by the Multi-domain Metalloenzyme SznF.

The alkylating warhead of the pancreatic cancer drug streptozotocin (SZN) contains an N-nitrosourea moiety constructed from Nω-methyl-l-arginine (l-NMA) by the multi-domain metalloenzyme SznF. The enzyme's central heme-oxygenase-like (HO-like) domain sequentially hydroxylates Nδ and Nω' of l-NMA. Its C-terminal cupin domain then rearranges the triply modified arginine to Nδ-hydroxy-Nω-methyl-Nω-nitroso-l-citrulline, the proposed donor of the functional pharmacophore. Here we show that the HO-like domain of SznF can bind Fe(II) and use it to capture O2, forming a peroxo-Fe2(III/III) intermediate. This intermediate has absorption- and Mössbauer-spectroscopic features similar to those of complexes previously trapped in ferritin-like diiron oxidases and oxygenases (FDOs) and, more recently, the HO-like fatty acid oxidase UndA. The SznF peroxo-Fe2(III/III) complex is an intermediate in both hydroxylation steps, as shown by the concentration-dependent acceleration of its decay upon exposure to either l-NMA or Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA). The Fe2(III/III) cluster produced upon decay of the intermediate has a small Mössbauer quadrupole splitting parameter, implying that, unlike the corresponding product states of many FDOs, it lacks an oxo-bridge. The subsequent decomposition of the product cluster to one or more paramagnetic Fe(III) species over several hours explains why SznF was previously purified and crystallographically characterized without its cofactor. Programmed instability of the oxidized form of the cofactor appears to be a unifying characteristic of the emerging superfamily of HO-like diiron oxidases and oxygenases (HDOs).

An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin.

Small molecules containing the N-nitroso group, such as the bacterial natural product streptozotocin, are prominent carcinogens1,2 and important cancer chemotherapeutics3,4. Despite the considerable importance of this functional group to human health, enzymes dedicated to the assembly of the N-nitroso unit have not been identified. Here we show that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. This biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N-N bond formation. We find that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria-including environmental organisms, plant symbionts and human pathogens-which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites.