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 04:00:00 UTC 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 Laboratory (ANL), Argonne, IL (United States)

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

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1771389

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

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Additional file 1 of Characterization of an efficient N-oxygenase from Saccharothrix sp. and its application in the synthesis of azomycin Fan, Chuanle; Zhou, Fang; Huang, Wei figshare https://doi.org/10.6084/m9.figshare.24847098.v1	image	January 2023

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59 BASIC BIOLOGICAL SCIENCES
N-oxygenase
biochemistry
bioinformatics
biological sciences
biophysics and computational biology
physical sciences
streptozotocin
x-ray crystallography

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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