An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin

Abstract

N-nitroso-containing small molecules, such as the bacterial natural product streptozotocin, are prominent carcinogens 1,2 and important cancer chemotherapeutics 3,4 . Despite this functional group's significant impact on human health, dedicated enzymes involved in N-nitroso assembly have not been identified. Here, we describe a metalloenzyme from streptozotocin biosynthesis (SznF) that catalyzes an oxidative rearrangement of the guanidine group of N ω -methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF uncovered two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. The discovery of this biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-heme iron-dependent enzymes to include N-N bond formation. We find biosynthetic gene clusters encoding SznF homologs are widely distributed among bacteria, including environmental organisms, plant symbionts, and human pathogens, suggesting an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites. Streptozotocin (SZN) (Zanosar®) is an N-nitrosourea natural product and approved cancer chemotherapeutic (Fig. 1a) 3,5 . SZN is also used to induce type I diabetes in animal models due to its toxicity towards pancreatic beta cells (> 28,500 PubMed references) 6 . Like other N-nitrosoureas, SZN exerts its activity in vivo by generating electrophilic DNA alkylating Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://

In vitro Reconstitution of the Biosynthetic Pathway to the Nitroimidazole Antibiotic Azomycin

“…We also observed strict iron-dependence for the reaction (Figure 4a and Supporting Information, Figure S15). [25,26] We also obtained Michaelis-Menten kinetic parameters for the KAzRohS-catalyzed oxidation of 5,d etermining a K M of 214 mm,a nd a k cat of 0.314 min À1 (Supporting Information, Figure S16). [25,26] We also obtained Michaelis-Menten kinetic parameters for the KAzRohS-catalyzed oxidation of 5,d etermining a K M of 214 mm,a nd a k cat of 0.314 min À1 (Supporting Information, Figure S16).…”

“…[22,23] Furthermore,t he Rieske oxygenase PrnD employs aR ieske [2Fe-2S] cluster and mononuclear iron center to catalyze an aryl-amine oxidation. [25,26] We constructed as equence alignment between RohS,k nown aryl-amine oxygenases,a nd the diiron domain of StzF.W hile there is limited overall homology between RohS and the aryl-amine oxygenases, the putative diiron binding sites in StzF are closely related to those in RohS (Supporting Information, Figure S14). This domain hydroxylates the guanidium nitrogens of the arginine-derived compound N w -monomethyll-arginine.…”

“…Thei nclusion of RohT did not alter the amount of 6 produced by KAzRohS,a nd RohT could not catalyze the oxidation of 5 to 6 under these conditions (Supporting Information, Figures S19 and S20). [26] Despite our inability to reconstitute the activity of RohT in vitro,t he four enzymes characterized should be sufficient for 6 production in ao ne-pot reaction. A similar lack of in vitro activity was also reported for the related Rieske ferredoxin from the streptozocin biosynthetic pathway.…”

“…This domain hydroxylates the guanidium nitrogens of the arginine-derived compound N w -monomethyll-arginine. [25,26] We constructed as equence alignment between RohS,k nown aryl-amine oxygenases,a nd the diiron domain of StzF.W hile there is limited overall homology between RohS and the aryl-amine oxygenases, the putative diiron binding sites in StzF are closely related to those in RohS (Supporting Information, Figure S14).…”

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

“…To exclude the possibility of polar effects caused by the gene replacements,w ec hemically complemented the mutants with synthetic l-graminine (Gra) that would be activated by the respective adenylation domains and transformed into the enantiomeric building block by the adjacent epimerase domains.W ef ound that the supplement restored gramibactin production in both mutants ( Figure 1B), Thus, grbD and grbE were proven essential for graminine biosynthesis.This finding is intriguing since the closest characterized homologue of GrbD,S znF,h as just been shown to mediate the nitrosylation of au rea moiety in streptozotocin biosynthesis. [13] Whereas GrbE may play ar ole in the formation of the hydroxylamine,G rbD apparently mediates NÀN-bond formation. This strategy is markedly different from the fragin biosynthetic pathway,w here an AurF-like enzyme has been implicated in nitroso formation.…”