Nosiheptide biosynthesis featuring a unique indole side ring formation on the characteristic thiopeptide framework

Abstract

Nosiheptide (NOS), belonging to the e series of thiopeptide antibiotics that exhibit potent activity against various bacterial pathogens, bears a unique indole side ring system and regiospecific hydroxyl groups on the characteristic macrocyclic core. Here, cloning, sequencing, and characterization of the nos gene cluster from Streptomyces actuosus ATCC 25421 as a model for this series of thiopeptides has unveiled new insights into their biosynthesis. Bioinformatics-based sequence analysis and in vivo investigation into the gene functions show that NOS biosynthesis shares a common strategy with recently characterized b or c series thiopeptides for forming the characteristic macrocyclic core, which features a ribosomally synthesized precursor peptide with conserved posttranslational modifications. However, it apparently proceeds via a different route for tailoring the thiopeptide framework, allowing the final product to exhibit the distinct structural characteristics of e series thiopeptides, such as the indole side ring system. Chemical complementation supports the notion that the S-adenosylmethionine-dependent protein NosL may play a central role in converting tryptophan to the key 3-methylindole moiety by an unusual carbon side chain rearrangement, most likely via a radical-initiated mechanism. Characterization of the indole side ring-opened analogue of NOS from the nosN mutant strain is consistent with the proposed methyltransferase activity of its encoded protein, shedding light into the timing of the individual steps for indole side ring biosynthesis. These results also suggest the feasibility of engineering novel thiopeptides for drug discovery by manipulating the NOS biosynthetic machinery.

Figure 1

Structures of the e series thiopeptides nosiheptide (NOS) and nocathiacin I, b series thiostrepton (TSR)/siomycin A (SIO-A), and d series thiomuracin A (TMR-A), GE2270A and thiocillin I (TCL-I).

Figure 2

Organization of the NOS biosynthetic genes. The deduced functions of which are labeled in color and summarized in Table 1.

Figure 3

Genetic features for the thiopeptide framework formation. (a) Peptide precursors for NOS (NosM), TSR (TsrH), SIO-A (SioH), TCLs (TclB), and GE2270A (TpdA). The SP sequences are labeled in bold. (b) Organization of the thiopeptide framework-forming genes identified from the producers of NOS (nos), TSR (tsr), SIO-A (sio), and TCLs (tcl). Their deduced functions are labeled in pattern, and homologies in sequence are indicated by dashed lines.

Figure 4

Proposed biosynthetic pathway of the indolic acid moiety of NOS.

Figure 5

Biosynthetic hypothesis for conversion of the thiopeptide framework into NOS. Color coding indicates the posttranslational modifications on the peptide precursor NosM (orange), attachment of the indolic acid moiety to the macrocyclic core (purple), and tailoring steps including hydroxylations on Glu43 at the γ-position and central pyridine domain at the C-5 position, and final cleavage of an acrylic acid to afford NOS (blue).

Figure 6

HPLC analysis of the production of NOS or its analog, in the wild type strain S. actuosus ATCC 25421 (I), nosM mutant SL4002 (II), recombinant strain SL4003 that derives from SL4002 by expressing nosM in trans (III), nosH mutant SL4004 (IV), nosL mutant SL4005 (V), nosL mutant SL4005 complemented by chemically feeding 3-methylindole-2-carboxylic acid (8) (VI), nosN mutant SL4006 (VII), and nosP mutant SL4007 (VIII). Solid dot indicates NOS. Solid triangle indicates the NOS analog 4.

Figure 7

MS/MS spectrum of the [M + Na]⁺ ion at m/z = 1269.74 corresponding to the NOS analog 4. The obtained fragments clearly indicate a permutation and combination pattern (labeled in color) for cleavage on the free carboxylic group(s) of the C-terminal dehydroalanine acid and/or side chain of Glu43, and/or on the linkage of the 3-methylindole moiety (2) to Cys45 of the macrocyclic core.