Variation in biosynthesis and metal-binding properties of isonitrile-containing peptides produced by Mycobacteria versus Streptomyces

Abstract

A number of bacteria are known to produce isonitrile-containing peptides (INPs) that facilitate metal transport and are important for cell survival; however, considerable structural variation is observed among INPs depending on the producing organism. While non-heme iron 2-oxoglutarate dependent isonitrilases catalyze isonitrile formation, how the natural variation in INP structure is controlled and its implications for INP bioactivity remain open questions. Herein, total chemical synthesis is utilized with X-Ray crystallographic analysis of mycobacterial isonitrilases to provide a structural model of substrate specificity that explains the longer alkyl chains observed in mycobacterial versus Streptomyces INPs. Moreover, proton NMR titration experiments demonstrate that INPs regardless of alkyl chain length are specific for binding copper instead of zinc. These results suggest that isonitrilases may act as gatekeepers in modulating the observed biological distribution of INP structures and this distribution may be primarily related to differing metal transport requirements among the producing strains.

Figure 1.

(A) Structures of INPs show that they are composed of different peptide backbone and NC moieties. According to tandem-MS metabolic analysis, 3 is composed of a mixture of compounds differing in the length of the alkyl group. (B) Isonitrilase catalyzed isonitrile formation involves desaturation and decarboxylation (6 → 7 → 8). The β-NC-containing acid (8) is then introduced to the peptide backbone via an NRPS to form the INP. The present work reveals that two isonitrilases from mycobacteria utilize the free carboxylic acid with (R)-chirality as the substrate as opposed to (S)-6, heptyl-(R)-6-SNAc or ACP-6.

Figure 2.

(A) Isonitrilase assays involved incubating 120 uM enzyme and 100 uM ferrous iron with 1 mM substrate and 2 mM 2OG at pH 7.5 and 4 °C for 12 h before workup with tetrazine to generate pyrazole. (B) Rv0097 and MmaE show significantly less isonitrile formation using heptyl-(S)-6 and heptyl-(R)-6-SNAc versus heptyl-(R)-6 as the substrate. (C) Relative extent of isonitrile formation establishes that both Rv0097 and MmaE prefer substrates bearing an alkyl chain ranging in length from 5 to 11 carbons with undecyl-(R)-6 (n = 11) being the optimal substrate.

Figure 3.

Active sites of (A) Rv0097 with heptyl-6 (yellow), PDB ID: 8KHT, (B) MmaE with heptyl-6 (yellow), PDB ID: 8KIF, and (C) ScoE with methyl-6 (pink), PDB ID: 6L6X. 2Fo-Fc (light gray mesh, contoured at 1.0 σ) electron density map for methyl-6. Dashed lines illustrate distances between the glycine α-carbon and iron (orange sphere). (D) Superposition of ScoE•Fe•methyl-6 and Rv0097•Fe•heptyl-6 ternary complexes. Rv0097 is colored in cyan and ScoE is colored in gray. Methyl-6 (pink) and heptyl-6 (yellow) are nearly superimposed in the enzyme active site.

Figure 4.

Total synthesis of INP 1, 2, 3 and 9. Reagents and conditions: (a) dodecanal, tert-butyl 2-(triphenylphosphoranylidene)acetate, THF, 0 to 23 °C, 87%. (b) (R)-(+)-N-Benzyl-1-phenylethylamine, n-butyllithium (n-BuLi), THF, ‒78 °C, 69%. (c) (i) Trifluoroacetic acid (TFA), DCM, 23 °C; (ii) Pd/C, MeOH, H₂, 23 °C, 71% over 2 steps. (d) (i) Trimethylsilane (TMSCl), MeOH, 0 to 23 °C; (ii) ethyl formate, 55 °C; (iii) lithium hydroxide (LiOH), THF/H₂O, 23 °C, 38% over 3 steps. (e) (i) N-Hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), THF, 0→23 °C; (ii) Burgess reagent, CHCl₃, 65 °C, 69% over 2 steps. (f) 15/17/18, trimethylamine (Et₃N), THF, 0 to 23 °C. (g) tetra-n-butylammonium fluoride (TBAF), THF, 0 to 23 °C. (h) K₂CO₃, MeOH/H₂O, 23 °C, 79%. Compounds 1, 2 and 9 were prepared analogously from 10.

Figure 5.

¹H-NMR titration of NC-peptides (1, 2, or 3) with Zn(II) or Cu(I). (A) No peak shift was detected in the presence of Zn(II), suggesting INPs do not bind to zinc. (B, C and D) Peak shifts were observed upon the addition of Cu(I) to a solution containing 1, 2 or 3. The β-proton resonances of the NC moieties are highlighted in red, whereas the δ- or ε-proton resonances of the peptide backbone are highlighted in blue.