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

Abstract

In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain 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 mono-iron cofactor in the enzyme’s C-terminal cupin domain, which effects 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. Here we leverage our recent report of an intensely absorbing µ-peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2 to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2 by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-like N-oxygenase but not in two known HO-like diiron desaturases. Among ∼9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, ∼25% have this carboxylate residue and are thus tentatively assigned as N-oxygenases.Significance statementThe enzyme SznF assembles the N-nitrosourea pharmacophore of the drug streptozotocin. Its central N-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. We investigated assembly of the O2-reactive diiron complex from metal-free SznF and Fe(II) and leveraged this insight to obtain the first structure of a functionally assigned HDO with intact cofactor. Conformational changes accompanying cofactor acquisition explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of the HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs.

CADD from Chlamydia trachomatis is a manganese-dependent oxygenase that employs a self-sacrificing reaction for the synthesis of p-aminobenzoate

“…As-purified, CADD contained ~0.05-0.1 Fe ions per monomer, indicating that the majority of the enzyme population lacks the expected diiron cofactor. This result is consistent with other HDO family members (6,7,9,12,13,17), where the metallocofactor is apparently highly unstable. LC-MS analysis of reactions containing as-purified CADD (154 µM) under aerobic conditions showed the production of ~3 µM pAB when the enzyme was incubated with dithiothreitol (DTT) versus ~1 µM pAB detected when incubated without a reducing agent (Figure 2A).…”

“…Thus, CADD is a member of the emerging heme-oxygenase-like diiron oxidase (HDO) superfamily, of which ~10,000 members are bioinformatically proposed, but only a handful have defined enzymatic activities (6). Characterized HDOs catalyze diverse reactions including N-oxygenation (SznF (6)(7)(8)(9), RohS (10), and FlcE (11)), oxidative C-C bond cleavage (UndA (12)(13)(14), BesC (15)(16)(17), and FlcE (11)), and methylene excision (FlcD) (11).…”

Nitrogen–Nitrogen Bond Formation Reactions Involved in Natural Product Biosynthesis

“…It has been proposed that the NO generated in situ could bind to the active site iron of the StzF cupin domain to form a nitrosyl-metal intermediate and then be incorporated into L-HMC to provide an N -nitroso group . The fact that no N -nitroso product was observed in the StzF in vitro reactions might due to the very little iron bound (<0.02 equiv) by StzF and the instability of di-iron cofactor as described elsewhere. , NO is an active species for nitrogen–nitrogen bond formation involved in both the inorganic nitrogen cycle and natural product biosynthesis. , The nitrosyl-metal intermediate has already been identified in the reaction catalyzed by NO reductase CYP450nor, which converts NO to N 2 O (Figure C) . Taken together, we cannot rule out the possibility that NO is the distal nitrogen atom donor of the N -nitroso group in streptozotocin ( 5 ) biosynthesis.…”

“…62 The fact that no N-nitroso product was observed in the StzF in vitro reactions might due to the very little iron bound (<0.02 equiv) by StzF and the instability of di-iron cofactor as described elsewhere. 63,64 NO is an active species for nitrogen−nitrogen bond formation involved in both the inorganic nitrogen cycle and natural product biosynthesis. 25,39 The nitrosyl-metal intermediate has already been identified in the reaction catalyzed by NO reductase CYP450nor, which converts NO to N 2 O (Figure 2C).…”

“…Alternatively, the other proposal confirmed that SznF di-iron domain sequentially hydroxylates two nitrogen atoms of the guanidine group of 25 to form L-DHMA (28; Figure 4 A). 61,63,64 The rearrangement of L-DHMA (28) to form nitrogen−nitrogen bond occurs in the SznF cupin domain in which the iron is coordinated to three His residues (H448, H409 and H307). The coordination of the dihydroxyl intermediate 28 with SznF indicates a significant binding pattern between the N δ -hydroxyl moiety and the iron center.…”