Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF.

In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω' of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme's C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability-a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon-hydrogen and carbon-carbon bonds to install olefins. Among ∼9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, ∼25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases.

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).

Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis.

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.