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

Abstract

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.

Biosynthesis of fosfomycin in pseudomonads reveals an unexpected enzymatic activity in the metallohydrolase superfamily

“…Our findings resemble the discovery that the HD domain family of enzymes, which were presumed to be metalloenzymes with phosphohydrolase activity, encompass members with iron-dependent oxygenase activity (49)(50)(51)(52). The discovery of an activity of a PHP family member that is very different from previously reported activities, as well as the phylogenetic analysis, suggests that this enzyme superfamily may catalyze a much larger set of reactions than previously appreciated and adds to the known folds that support dinuclear iron chemistry (32,34,53,54).…”

“…The distance between the two Fe atoms is 4.9 Å, which is considerably longer than the 2.5 to 3.4 Å distances observed in canonical nonheme diiron enzymes (SI Appendix, Table S1) (32). However, similar long Fe-Fe separation distances have been reported in the structures of diiron-reconstituted UndA (33) and SznF (34). In the case of UndA, binding of the substrate triggers the formation of a μ-peroxo-Fe(III/III) with an Fe-Fe separation of 3.21 Å, as determined by extended X-ray absorption fine structure studies.…”

A Substrate-triggered µ-Peroxodiiron(III) Intermediate in the 4-Chloro-L-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate

“…1 A strategy of (i) preparing naive apo protein by heterologous expression in low-iron medium supplemented with Mn(II) to occupy the metal sites and prevent potentially deleterious cofactor cycling during protein production, (ii) chelation of the bound Mn(II) from the purified protein, (iii) crystallization of the apo protein, and (iv) soaking of crystals with Fe(II) yielded a structure of the SznF•Fe(II) 2 complex with both sites almost fully occupied, the first such structure of an HDO. 26 Comparison of apo and metallated structures revealed that one of the iron sites in the HDO domain (site 2) is not pre-formed (Fig. S1), by contrast to observations made on structures of apo FDOs.…”

“…This dichotomy within the first two mechanistically characterized HDOs mirrors that seen in the FDOs, of which the N-oxygenases AurF and CmlI were shown not to require triggering by either the substrate or an accessory protein. 24 Recent x-ray crystallographic studies of SznF 26 shed light on the instability of the diiron cofactor in its HDO domain, 19 and structural characteristics shared with UndA 18,23 suggested that the insight might be general to other (potentially all) enzymes in the emerging HDO family. 1 A strategy of (i) preparing naive apo protein by heterologous expression in low-iron medium supplemented with Mn(II) to occupy the metal sites and prevent potentially deleterious cofactor cycling during protein production, (ii) chelation of the bound Mn(II) from the purified protein, (iii) crystallization of the apo protein, and (iv) soaking of crystals with Fe(II) yielded a structure of the SznF•Fe(II)2 complex with both sites almost fully occupied, the first such structure of an HDO.…”

“…[27][28][29][30] An 8-residue segment of the protein was seen to rearrange from a series of turns in the apo state to a canonical a-helix (part of a3) in the diiron(II) complex, in the process relocating a site 2 carboxylate ligand (D315) ~10 Å from the protein surface into the helical core. 26 It was posited that dynamics of the modestly stable a3 could initiate the observed disintegration of the product diiron(III) complex. Given that a segment of a3 in UndA could not even be confidently modeled because of poor (or absent) electron density, 23 this explanation for cofactor instability could be applicable also to the founding HDO.…”