N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry

Abstract

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

Fig. 1. Chemical structures of STs and ST-related antibiotics.

a The gene product of orf1 encoded in the BD-12 producing strain is able to catalyze glycinothricin to BD-12. Other than glycinothricin, glycylthricin, 3-aminopropionylthricin, 4-aminobutylthricin and ST-F can serve as substrates of Orf1 to bring on N-formimidoylated or N-iminoacetylated corresponding products. b The N-formimidoylation or N-iminoacetatylation was presumed to follow multiple steps of reactions in one single reaction site as does a typical FAD-dependent enzyme. c The proposed mechanisms of N-formimidoylation or N-iminoacetylation catalyzed by Orf1 that recruits a FAD prosthetic group and an NOS protein-ligand covalent modification to enable multiple reactions to take place at two separate but adjacent reaction chambers. Four-membered peroxide and three-membered oxaziridine that may respectively react with oxidized and non-oxidized cysteine to form the NOS linkage, however, were not detected in any circumstances. In the presence of proper acceptors (e.g., 1 or 4), products 2, 5 with the N-formimidoyl modification are formed by Orf1, in which sulfenic C281 is recyclable (reactivated only once). R’HN’ colored blue is glycylthricin analogs (e.g., compound 7) with various aliphatic side chains in length leading to N-iminoacetyl modification. In this circumstance, apart from hydrogen peroxide (H₂O₂) sulfenic C281 can be regenerated by water as determined by isotope labeling experiments using H₂¹⁸O highlighted red (see Fig. 5c).

Fig. 2. The biochemical examinations for Orf1 and mutants thereof against glycylthricin and ST-F.

a The EIC traces of formimidoyl-glycylthricin (Fig. 1a, compound 5) (m/z 459.2 [M + H]⁺) for reactions catalyzed by (i) denatured Orf1, (ii) Orf1, (iii) C281A, (iv) C281S, (v) E312A, or (vi) F316A. b Mass spectra of 5 produced in the Orf1-mediated reactions in the presence of 4 and (i) glycine ([M + H]⁺ = 459), (ii) ¹⁵N-glycine ([M + H]⁺ = 460), (iii) 1-¹³C-glycine ([M + H]⁺ = 459), or (iv) 2-¹³C-¹⁵N-glycine ([M + H]⁺ = 461). c Mass spectra of iminoacetyl-glycylthricin (Fig. 1a, compound 6) produced in the Orf1-mediated reactions in the presence of 4 and (i) glycine ([M + H]⁺ = 503), or (ii) ¹³C₂-¹⁵N-glycine ([M + H]⁺ = 506). d The EIC traces of ST-F (Fig. 1a, compound 7) (m/z 503.2 [M + H]⁺) and iminoacetyl-ST-F (Fig. 1a, compound 8) (m/z 574.2 [M + H]⁺) for reactions catalyzed by (i) denatured Orf1, (ii) Orf1, (iii) Orf1 with addition of DTT, (iv) C281S, (v) R342A, (vi) C281S and R342A, (vii) ThiO and R342A, (viii) E426Q, (ix) E312A, or (x) F316A. e Mass spectra of the Orf1-mediated reactions in the presence of ST-F and (i) glycine ([M + H]⁺ = 574), (ii) ¹⁵N-glycine ([M + H]⁺ = 575), (iii) 1-¹³C-glycine ([M + H]⁺ = 575), (iv) 2-¹³C-¹⁵N-glycine ([M + H]⁺ = 576), or (v) ¹³C₂-¹⁵N-glycine ([M + H]⁺ = 577). Source data are provided as a Source Data file.

Fig. 3. Overall structures of Orf1.

a Orf1 is a tetrameric complex of a dimer of dimers in solution, of which each protomer contains a FAD prosthetic group. b Each protomer is comprised of three domains, a FAD binding domain (red), a substrate binding domain (green) and a C-terminal domain (blue). The tetrameric complex is interfaced by the substrate binding domain and the C-terminal domain. c The glycine binding site is located on the top of the re face of the isoalloxazine ring of FAD and shaped by residues A54, G55, A56, M57, Y294, H284, R342, and R368 revealed from the Orf1-glycine complex. d Sarcosine also binds to the glycine-binding site as revealed from the Orf1-sacrosine complex. e R368 is misplaced when R342 was mutated to Ala resulting in the loss of enzyme activity as revealed from the Orf1-R342A structure. f The surface presentation of the glycine binding pocket, where a cavity nearby the active site was speculated the glycylthricin binding site. g The complex structure of Orf1-glycine-glycylthricin. Each Orf1 protomer in the tetrameric complex contains one molecule of FAD, glycine and glycylthricin. h The glycylthricin binding site is located at the inter-subunit junctions, where binding pockets are formed. i The 2F_o-F_c density map of glycylthricin is countered at 1 σ shown in a gray mesh. j The distance between the amine nitrogen atom of glycylthricin and the α-carbon of glycine is 13.5 Å away from each other. The antiparallel-β-sheet (β11- β15) acts as a physical barrier that delimits a boundary for both the glycylthricin and glycine binding pockets. k Superposition of crystal structures of wildtype and mutants in complex with glycylthricin. The Orf1-glycylthricin (B chain), C281S-glycylthricin (B chain), F316A-glycylthricin (B chain), R342A-glycylthricin (A chain) and E426Q-glycylthricin (B chain) are colored green, cyan, gray, magentas and yellow, respectively. The major binding-site residues and glycylthricin are well aligned without significant conformational changes.

Fig. 4. The glycine imine-N–O–S-C281 bridge in the Orf1-glycine complex structure.

a An intrinsic electron density (contrasted by 2F_o-F_c map contoured at 1 σ in gray and the F_o-F_c map contoured at 3 σ in green) was unexpectedly found nearby residue C281 at the glycylthricin binding site. b The chemical model that best fits into the electron density region (2F_o-F_c map at 1 σ colored blue) is a glycine imine adduct in a covalent linkage to C281 through a N–O–S bridge. c The C281-iminoglycine adduct is surrounded by residues T298, E312, E313, F316 and E426. The distance between two atoms was labeled with a yellow dashed line. d, e Superposition of two complexes (the C281-adduct and the glycylthricin-containing structures) shows that the distance between the amine group of glycylthricin and α-carbon of the iminoacetate adduct is 3.1 Å within a general H-bond range with a Burgi-Dunitz angle of 99.7° in an approaching trajectory. f Superposition of WT-glycylthricin (green), E312A-glycylthricin (cyan) and C281-iminoglycine adduct (gray) reveals that E312 undergoes a substrate-induced conformational change, in which upon addition the resulting decarboxylation facilitates β-elimination. g Superposition of WT-glycylthricin (green), WT-4-aminobutylthricin (magenta), F316-ST-F (cyan) and C281-iminoglycine adduct (gray) shows considerable displacements of the median γ-aminobutyl and the long β-lysine sidechains away from that of glycylthricin likely as a result of a geographic effect.

Fig. 5. The determination of covalent modifications of residue C281.

a Mass spectrometric analyses for the C281-dimedone adduct and the sulfinylated peptide fragments of Orf1 incubated in a glycine-containing buffer solution. Detection of the AFAC²⁸¹GLHLVPR peptide labeled with dimedone (+138 Da) and the sulfinylated peptide (+33 Da). MS/MS spectrum indicates the formation of a C281-dimedone adduct confirming the existence of the SOH group. The mass spectrometric analysis concurrently revealed that the thiol can undergo stepwise oxidation to sulfenic, sulfinic and sulfonic acid (Supplementary Fig. 29). The conversion rate for SH-Cys281 to SOH-Cys281 is fast and high. The mass spectrometry files were deposited in ProteomXchange with identifier PXD041105 and PXD041104, respectively. b A considerable amount of H₂O₂ detected in the glycine-containing reaction solution added with ThiO (FPOX) in 3 min in contrast to much less H₂O₂ in the same context but added with Orf1 (FPMO). The assays of Orf1 and ThiO were run with two independent replicates (n = 2) and the data were presented as mean values ± SD. Source data are provided as a Source Data file. Photospectrometric analysis further supports Orf1 a FPMO (Supplementary Fig. 30). c The sulfonic-containing peptides (at C281) from trypsin-treated Orf1 were selected and analyzed by mass spectrometry, in that the sulfonic-containing fragment renders relatively high abundance with molecular distinctiveness. Orf1 was incubated in the presence of 4 or 7 in a H₂¹⁸O buffer solution 1 h and then subjected to trypsin digestion overnight, of which the mass spectra demonstrated that the incorporation of one ¹⁸O-isotopic atom in sulfonic-containing fragments occurs mainly in the reaction with 7 (lower panel, with an enriched M + 2 peak) as opposed to that with 4 (upper panel, with a typical isotopic profile) in favor of the mechanism proposed in Fig. 1c. z = 2 stands for doubly-charged positive ions in mass spectrometry. The reaction condition is the same as that for the experiments shown in Supplementary Fig. 31c(ii) and Supplementary Fig. 9e(ii). Two major products are 5 and 8, respectively. The mass spectrometry files were deposited in ProteomXchange with identifier PXD041103.