Genomics‐Driven Discovery of NO‐Donating Diazeniumdiolate Siderophores in Diverse Plant‐Associated Bacteria

Hermenau, Ron; Mehl, Jule L; Ishida, Keishi; Dose, Benjamin; Pidot, Sacha J.; Stinear, Timothy P.; Hertweck, Christian

doi:10.1002/anie.201906326

Cited by 57 publications

(93 citation statements)

References 36 publications

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“…At least four HO-like diiron enzymes (HDOs) -a pair of N-oxygenases (37)(38)(39)(40) and a pair of olefin-forming lyase/oxidase (hereafter, desaturase) enzymes (41-44) -have been described to date, and several others have been identified within biosynthetic gene clusters ( Fig. S2) (45)(46)(47)(48). The presumptive enzymatic activity of the first structurally characterized protein in this new superfamily, Chlamydia protein associated with death domains (CADD), is still unknown (49,50).…”

Section: Significance Statementmentioning

confidence: 99%

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

McBride¹,

Pope

Kai

et al. 2020

Preprint

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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.

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Section: Significance Statementmentioning

confidence: 99%

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

McBride¹,

Pope

Kai

et al. 2020

Preprint

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“…HamC, which has been demonstrated to oxidize p ‐aminobenzoic acid to p ‐nitrobenzoic acid in vitro, is proposed to mediate N−N bond formation in fragin biosynthesis, but this putative reaction has yet to be verified in vivo and in vitro. Similarly, the SznF homologue GrbD from the gramibactin pathway is proposed to catalyze N−N bond formation, but its activity has not been demonstrated . Elucidating the biosynthesis of 5 would therefore improve our limited insights into enzymatic installation of the diazeniumdiolate, and more broadly the N ‐nitroso group.…”

Section: Figurementioning

confidence: 99%

The l‐Alanosine Gene Cluster Encodes a Pathway for Diazeniumdiolate Biosynthesis

McCallum

Zheng

et al. 2019

ChemBioChem

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N‐Nitroso‐containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N‐nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l‐alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N‐nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non‐proteinogenic amino acid l‐2,3‐diaminopropionic acid (l‐Dap) is synthesized and loaded onto a free‐standing peptidyl carrier protein (PCP) domain in l‐alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.

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“…This compound was shown to possess antifungal, antitumour, antibacterial, and plant growth inhibitory activities . More recently, the previously unknown signaling compound rac ‐valdiazen ( 2 ) was isolated from Burkholderia cenocepacia H111 (H111), and in the same year Hertweck and co‐workers discovered a new family of diazeniumdiolate siderophores …”

Section: Methodsmentioning

confidence: 99%

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

et al. 2020

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Only a few natural products incorporating a diazeniumdiolate moiety have been isolated, and these compounds usually display a broad range of biological activities. Only recently has the first diazeniumdiolate natural product biosynthetic gene cluster been identified in Burkholderia cenocepacia H111, which produces the fungicide (−)‐fragin and the signal molecule rac‐valdiazen. In this study, l‐valine was identified as the initial substrate of (−)‐fragin biosynthesis with the aid of feeding experiments using isotopically labelled amino acid. The formation of the diazeniumdiolate was chemically studied with several proposed intermediates. Our results indicate that the functional group is formed during an early stage of the biosynthesis. Furthermore, an oxime compound was identified as a degradation product of (−)‐fragin and was also observed in the crude extract of the wild‐type strain. Moreover, a structure–activity relationship analysis revealed that each moiety of (−)‐fragin is essential for its biological activity.

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Genomics‐Driven Discovery of NO‐Donating Diazeniumdiolate Siderophores in Diverse Plant‐Associated Bacteria

Cited by 57 publications

References 36 publications

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

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

The l‐Alanosine Gene Cluster Encodes a Pathway for Diazeniumdiolate Biosynthesis

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

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