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

McBride, Molly J.; Pope, Sarah R.; Hu, Kai; Okafor, C. Denise; Balskus, Emily P.; Bollinger, Jr., J. Martin; Boal, Amie K.

doi:10.1073/pnas.2015931118

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

Journal Article · Tue Jan 19 00:00:00 EST 2021 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2015931118· OSTI ID:1771389

^[1];

^[1]; Okafor, C. Denise ^[1]; Balskus, Emily P. ^[2]; Bollinger, Jr., J. Martin ^[1]; Boal, Amie K. ^[1]

Pennsylvania State Univ., University Park, PA (United States)
Harvard Univ., Cambridge, MA (United States)

In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates N^δ and N^ω' of N^ω-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.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Institutes of Health (NIH); Michigan Technology Tri-Corridor; National Cancer Institute (NCI); National Institute of General Medical Sciences

Grant/Contract Number:: AC02-06CH11357; S10 OD012289; GM119707; GM138580; 085P1000817; ACB-12002; AGM-12006

OSTI ID:: 1771389

Alternate ID(s):: OSTI ID: 1772784

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 118, Issue 4; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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59 BASIC BIOLOGICAL SCIENCES
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x-ray crystallography
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Title: Structure and assembly of the diiron cofactor in the heme-oxygenase–like domain of the N-nitrosourea–producing enzyme SznF

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