doi: 10.1038/s41586-019-0894-z. Epub 2019 Feb 6.
An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin
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- PMID:30728519
- PMCID: PMC6369591
- DOI: 10.1038/s41586-019-0894-z
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An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin
Tai L Ng et al. Nature.2019 Feb.
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Abstract
Small molecules containing the N-nitroso group, such as the bacterial natural product streptozotocin, are prominent carcinogens1,2 and important cancer chemotherapeutics3,4. Despite the considerable importance of this functional group to human health, enzymes dedicated to the assembly of the N-nitroso unit have not been identified. Here we show that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. This biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N-N bond formation. We find that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria-including environmental organisms, plant symbionts and human pathogens-which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites.
Figures
a, Previous feeding experiments suggested that D-glucosamine, L-citrulline or L-arginine, and L-methionine-derivedS-adenosylmethionine (SAM) would be building blocks for SZN biosynthesis.b, Mass spectra of culture extracts in which15N-nitrate,15N-nitrite, or15N-ammonium chloride were fed toS. achromogenes var. streptozoticus NRRL 2697. The expected masses ([M+H]+) for SZN,15N-SZN, [15N2]-SZN, and [15N3]-SZN are 266.0983, 267.0953, 268.0923, and 269.0894, respectively. These results contrast sharply with the strong labeling (>75%) observed in studies of pathways that use nitrite for diazo biosynthesis,.
a, Comparative genomic analysis ofS. achromogenes var. achromogenes NRRL B-2120, a nonproducer of SZN andS. achromogenes var. streptozoticus NRRL 2697 using Mauve. Theszn gene cluster is colored in red. Gene annotations are tabulated in Supplementary Table 1.b, Multiple sequence alignment of SznE with structurally characterized protein arginine methyl transferases (PRMT) from eukaryotes. Conserved residues involved in binding L-arginine are marked with an asterisk.c, Overlay of a homology model of SznE (green) with the crystal structure of PRMT7 fromT. brucei (PDB accession code 4M37) (orange). The highlighted carboxylate residues are involved in binding of the basic guanidine group.d, SDS-PAGE of purified SznE. The expected molecular weight is 40 kDa. Ladder = Precision Plus Protein All Blue Standards (BioRad).e, Mass spectra of SZN produced when feedingS. achromogenes var. streptozoticus NRRL withd3-L-NMA. The expected masses [M-H2O+H]+ for SZN andd3-SZN are 248.0993 and 251.1060, respectively.f, LC-MS traces demonstrating restoration of SZN production by the ΔsznE mutant upon chemical complementation with L-NMA. The extracted ion chromatograms (EIC) are generated within a 5 ppm window.
a, The mass spectrum of SZN [M-H2O+H]+ when 1 mM of [15N413C6]-L-arginine was added to fermentation culture. To determine whether the labeled SZN was a single or a mixture of isotopologues, degradation (panelb) and MS/MS (panelc) experiments were performed.b, Exposing SZN to UV-light generated a one-carbon and one-nitrogen labeled cyclic urea previously reported to be a denitrosated SZN product, indicating that the distal nitroso nitrogen is labeled.c, MS/MS fragmentation of SZN revealed a one-carbon labeled cyclic carbamate fragment, indicating that both theN-nitroso nitrogens are labeled.
Insertions of the antibiotic cassette into each of theszn biosynthetic genes were confirmed by PCR. Culture supernatant extracts from each mutant were analyzed with LC-HRMS. Extracted ion chromatograms for the amino acids ([M+H]+) were generated with a 5 ppm window.
a, SDS-PAGE of purified SznF and SznFG. The molecular weights of SznF and SznG are 54 kDa and 13 kDa, respectively. Ladder = Precision Plus Protein All Blue Standards (BioRad).b, Nitrite and nitric oxide (NO) were detected when Fe(II)-SznF and L-NMA were incubated together. Nitrite was detected with the Griess reagent, and absorbance was measured at 548 nm. Error bars represent standard deviation of the mean of three replicates. NO was trapped with Fe(II)-N-methyl-D-glucamine dithiocarbamate (MGD) and analyzed by electron paramagnetic resonance (EPR) spectroscopy at room temperature. Sodium 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) was used as a positive control for NO detection. Assays using [guanidino-15N2]-L-NMA as a substrate revealed changes in hyperfine splitting by EPR spectroscopy, indicating that NO is derived from the terminal guanidine group of L-NMA. We hypothesize that the NO detected derives from the degradation of3 or is generated as part of the N–N bond formation step.c, Comparison of retention times and MS/MS fragmentation patterns of1,2, Fmoc-3, and4 generated in SznF assay mixtures with the corresponding synthetic standards. NMR characterization and synthetic procedures are reported in the Supplementary Information.
a, When 1 mM L-arginine, 80 μM SznF, 20 μM phenazine methosulfate (PMS), and 5 mM NADH were incubated at room temperature for one hour, only trace amounts of a mass corresponding to L-hydroxyarginine (EIC ([M-H]-) = 189.0993) was observed. No masses corresponding to L-hydroxycitrulline (EIC ([M-H]-) = 190.0883), L-dihydroxyarginine (EIC ([M-H]-) = 205.0942), or the L-nitrosocitrulline (EIC([M-H])- = 203.0786) were observed.b, The [M-H]- mass spectrum of3 generated when [15N413C6]-L-NMA and unlabeled L-NMA were mixed in the same SznF reaction mixture.c, Testing the metal dependence of SznF. 80 μM ofapo-SznF was incubated with 200 μM of various divalent metals, 20 μM PMS, 1 mM L-NMA, and 5 mM NADH for one hour at room temperature. The EIC traces were generated with a 5 ppm window.d, O2 was rapidly consumed in the presence of L-NMA and SznF as measured by an optode. SznF E281A, which lacks a key predicted iron-binding residue in the central domain, failed to consume O2 above background. The background consumption of O2 arises from the nonenzymatic reduction of PMS by NADH.e, Incubating18O2, 1 mM L-HMA (1), and 80 μM SznF at room temperature for one hour resulted in labeling of two of theN-nitrosourea oxygens. MS/MS analysis revealed retention of theNδ-OH (data not shown).f, Addition of H218O to an SznF assay mixture did not label theN-nitrosourea group. The expected [M-H]- masses for Fmoc-3, [18O]-Fmoc-3, [18O2]-Fmoc-3, and [18O3]-Fmoc-3 are 455.1572, 457.1615, 459.1657, and 461.1700, respectively.g, Addition of catalase or superoxide dismutase to the assay mixtures did not affect SznF-catalyzedN-oxygenation as measured by the Griess assay. Error bars represent standard deviation of the mean of three replicates.
a, A diagram of the secondary structures found in the N-terminal domain (blue), central helical bundle domain (orange), and C-terminal cupin domain (green) of SznF. FeII ligands and proposed active site residues are indicated as black dots. Disordered regions are shown as dashed lines. The cupin FeII-binding site is depicted as a circle.b, An Fe anomalous difference map (orange mesh, contoured at 5.0σ) is shown for the fully-occupied mononuclear His-coordinated FeII site (orange sphere) in the cupin domain. Selected amino acids are shown in stick format.c, The central domain contains a partially occupied (~50%) Fe-binding site in selected crystals, with a smaller peak in the Fe anomalous difference map (orange mesh, contoured at 3.0σ).
a, SznF contains a large cavity (gray surface, 1.9 Å probe radius) in the middle of its central helical bundle domain (orange). Additionally, most of the secondary structures in this domain contain loop disruptions and disordered regions, suggesting considerable refolding upon binding/release of the L-NMA substrate and/or assembly of the iron-based cofactor.b, The central domain of SznF is similar in topology to heme oxygenase (HO), compared here to HO-2 fromSynechocystis sp. PCC 6803 (PDB accession code 1WOW). SznF contains an open pocket near the heme binding site in HO-2 but lacks conserved cofactor ligation and H-bonding motifs (panel d).c, SznF instead more closely resembles aChlamydia trachomatis dinuclear iron protein in this structural superfamily (CADD) implicated inpara-aminobenzoic acid biosynthesis. SznF conserves all of the metal-binding residues but fails to stably incorporate iron in this domain in the current preparations. All three systems share a propensity for distorted secondary structure motifs that perhaps enable complex formation with large and polar substrates for oxidative transformations.d, A structure-based sequence alignment of six HO-like enzymes in selected regions relevant to substrate/cofactor interaction and catalysis. SznF conserves all six His/carboxylate residues used to coordinate a dinuclear iron cluster in the active form of fatty acid oxidative decarboxylase UndA (PDB accession code 4WWZ) and in the uncharacterized CADD protein (PDB accession code 1RCW). Prior to our discovery and characterization of SznF, UndA was the only HO-like enzyme with a defined substrate and activity. The published crystal structure of UndA contains only a single iron ion and a mechanism was initially proposed utilizing a mononuclear cofactor (located in site 1). However, recent spectroscopic studies by Rajakovich et al. show that this enzyme uses a dinuclear non-heme iron cofactor and corresponding alternative reaction pathway. To date, the dinuclear form of UndA has remained refractory to crystallographic characterization due to a propensity for disorder in the helix containing the site 2 metal ion ligands. As in SznF, mutagenesis of any of the six predicted ligands to the recently characterized dinuclear site in UndA completely abolishes activity. As a consequence, we propose that all HO-like non-heme-iron proteins (including SznF) assemble a multinuclear cofactor but require a second protein or other factor to stabilize the active form in high yield and at high concentration.e, Comparative views of the cofactor site and/or substrate binding site in (left to right, top to bottom) SznF,C. trachomatis CADD,Pseudomonas fluorescens UndA,Klebsiella pneumoniae pyrroloquinoline quinone (PQQ) synthase PqqC (PDB accession code 1OTW),Synechocystis sp. PCC 6803 heme oxygenase (HO) 2 (PDB accession code 1WOW), andBacillus subtilis thiamin synthase TenA (PDB accession code 1YAK). Substrates, products, and selected side chains are shown in stick format. Iron ions and water molecules are shown as orange and red spheres, respectively.f, Additional mutation of the predicted iron-binding residues in the SznF central bundle helix domain abolishedN-oxygenation activity. Assay mixtures contained 1 mM L-NMA, 80 μM SznF or variant, 20 μM PMS, and 5 mM NADH and were incubated at room temperature for one hour. The EIC traces were generated with a 5 ppm window using the [M-H]- masses.
a, An extended water-mediated H-bonding network tethers the non-metabolizable ligand1 (green sticks, black lines) in the active site via its Me-Nω,Nδ-O(H), and backbone amine/carboxylate functional groups. Selected SznF amino acids are shown in stick format in panela and in gray lines in panelb. H-bonding/ionic interactions are shown as gray (panela) or blue (panelb) dashed lines. Analysis of the network suggests a mechanism for deprotonation of1 Me-Nω via Y459 and E98. The cupin active site also contains an open hydrophobic pocket near the unmethylatedNω position. Apart from the aforementioned Y459 interaction, there are no H-bonds between the substrate functional groups and residues in the active site.b, Ligand interaction map showing FeII-coordination interactions (distances in Å) and H-bonding interactions with selected sidechains and water molecules.c, Use of the substrate analogNω-hydroxy-Nω-methyl-L-arginine (5) at 1 mM final concentration with 80 μM SznF, 20 μM PMS, and 5 mM NADH resulted in production of only trace amounts of3 after incubation for one hour at room temperature. No [M-H]- masses corresponding to2,3 without anNδ-OH (EIC = 217.0942),4, or4 without anNδ-OH (EIC = 188.1041) were observed.d, The reaction catalyzed by the cupin domain does not require an external reductant. The conversion from2 to3 proceeded when 1 mM of2 was incubated with 80 μM SznF without NADH at room temperature for one hour. The EIC traces were generated with a 5 ppm window.
a, Maximum-likelihood phylogenetic tree inferred from 50 replicates showing the relationship between selected SznF homologs containing both the central domain and cupin domain (NCBI “non-redundant protein sequences” database, 2018) (e-value < 1E-50). The branch corresponding toS. achromogenes SznF is highlighted by a single asterisk. Bootstrap confidence values of >50 are indicated by black circles on the nodes. The amino acid sequence of UndA is used as an outgroup (highlighted as **). The sequences used to generate this tree are tabulated in Supplementary Table 2.b, Distribution of 352 SznF homologs that contain both a central domain and a cupin domain in different bacterial genera (IMG/JGI “all isolates” database, 2018) (e-value < 1E-5). See also Supplementary Table 3.c, Selected biosynthetic gene clusters encoding homologs of SznF.
a, The structures of selected FDA-approvedN-nitrosourea-containing drugs.b, Known strategies forN-nitrosation in biological systems. M = metalloenzyme.c, Feeding15N-nitrite to the SZN producer did not result in labeled SZN (Mass spectra inExtended Data Fig. 1b).d, Theszn biosynthetic gene cluster. Genes encoding DNA repair enzymes are highlighted in grey.e,1H NMR assay shows that SznE usesS-adenosyl-L-methionine (SAM) as a methyl donor to convert L-arginine toNω-methyl-L-arginine (L-NMA)in vitro.f, Gene inactivation studies of theszn gene cluster. Extracted ion chromatograms (EICs) for SZN. The two peaks correspond to the α- and β-anomers.g, Feeding labeled L-arginine to the producing organism suggests the transfer of the intact guanidine group to theN-nitrosourea. EIC ([M-H2O+H]+) for SZN = 248.0877, [15N]-SZN = 249.0847, [13C15N]-SZN = 250.0877, and [13C15N2]-SZN = 251.0847. See Extended Data Fig. 3 for assignment of the positions of carbon and nitrogen labels.
a, LC-MS time course of the SznF-mediated transformation of L-NMA toN-nitrosourea3. EICs are shown. The structures of1–4 were confirmed by comparison to synthetic standards (Extended Data Fig. 5c).b, LC-MS traces of SznFin vitro assays defining the requirements for activity. EICs are shown.c, Crossover experiment showed that SznF catalyzes rearrangement of an intact guanidine group. See Extended Data Fig. 6b for mass spectrum.d,18O labeling experiment revealed molecular O2 as the source of the three oxygen atoms inN-nitrosourea3.
a, SznF-derived intermediate1 complements thesznE mutant but not thesznF mutant. EICs for SZN ([M-H2O+H]+ = 248.0883) are shown.b, Proposed pathway for SZN biosynthesis.
a, The structure of the SznF homodimer, colored by domain, reveals two candidate active sites. Disordered regions are shown as black dashed lines. C-terminal FeII site is shown as an orange sphere and bound intermediate L-HMA (1) is shown in stick format. Predicted metal-binding residues are highlighted on the cartoon schematic at bottom.b, A mononuclear His-coordinated FeII site (orange sphere) in the cupin domain recruits intermediate L-HMA (green sticks) as a bidentate ligand. Selected amino acids are shown in stick format and a polder omit electron density map is shown in gray mesh and contoured at 3.0σ.c, The central domain lacks full occupancy metal ions in the crystals but conserves a series of His/carboxylate ligands found in related diiron proteins. See also Extended Data Fig. 7, 8.
To test the reactivity of the separate domains, L-NMA (panela),1 (panelb), or2 (panelc) were incubated with no enzyme, an SznF mutant with an inactive central domain (E215A), an SznF mutant with an inactive cupin domain (H407A H409A H448A), or WT SznF. EICs were generated within 5 ppm.d, The reactions catalyzed by the two metallocofactor binding sites of SznF.
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