Unveiling an indole alkaloid diketopiperazine biosynthetic pathway that features a unique stereoisomerase and multifunctional methyltransferase

Abstract

The 2,5-diketopiperazines are a prominent class of bioactive molecules. The nocardioazines are actinomycete natural products that feature a pyrroloindoline diketopiperazine scaffold composed of two D-tryptophan residues functionalized by N- and C-methylation, prenylation, and diannulation. Here we identify and characterize the nocardioazine B biosynthetic pathway from marine Nocardiopsis sp. CMB-M0232 by using heterologous biotransformations, in vitro biochemical assays, and macromolecular modeling. Assembly of the cyclo-L-Trp-L-Trp diketopiperazine precursor is catalyzed by a cyclodipeptide synthase. A separate genomic locus encodes tailoring of this precursor and includes an aspartate/glutamate racemase homolog as an unusual D/L isomerase acting upon diketopiperazine substrates, a phytoene synthase-like prenyltransferase as the catalyst of indole alkaloid diketopiperazine prenylation, and a rare dual function methyltransferase as the catalyst of both N- and C-methylation as the final steps of nocardioazine B biosynthesis. The biosynthetic paradigms revealed herein showcase Nature’s molecular ingenuity and lay the foundation for diketopiperazine diversification via biocatalytic approaches.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif

“…For comparison, the recently investigated methyltransferase NozMT from Nocardiopsis sp. CMB-M0232 (sharing 57% sequence identity with StspM1), which is known to be involved in the biosynthesis of nocardioazine B, does not accept either of these substrates …”

“…CMB-M0232 (sharing 57% sequence identity with StspM1), which is known to be involved in the biosynthesis of nocardioazine B, does not accept either of these substrates. 16 To elucidate the absolute configuration of the newly formed stereogenic centers in methylated LL-cWW 14 and DD-cWW 16, ROESY NMR spectra were measured for identification of spatially adjacent protons. For single-methylated DD-cWW 16, a correlation between Me-13 and the H-11 was observed, proving a relative configuration in which the H-11 proton and the Me-13 methyl group face toward the same side of the molecule.…”

“…15 Further investigations on the biosynthetic pathway have shown that starting from L-tryptophan as a natural amino acid, a cyclodipeptide synthase (CDPS) forms LL-cWW, which must be isomerized into its enantiomer DD-cWW by an isomerase prior to prenylation and the final methylation. 11,16 In comparison, lansai B (4), first isolated from Streptomyces sp. SUC1, an endophyte found on the aerial roots of Ficus benjamina, was proven to contain an L-tryptophan-derived structure.…”

“…Consequently, nocardioazine B ( 3 ) consists of an endo -pyrroloindole and an exo -pyrroloindole, which fits the macrocyclic structure of nocardioazine A ( 2 ) . Further investigations on the biosynthetic pathway have shown that starting from L - tryptophan as a natural amino acid, a cyclodipeptide synthase (CDPS) forms LL- cWW, which must be isomerized into its enantiomer DD - cWW by an isomerase prior to prenylation and the final methylation. , …”

“…Focusing on the synthesis of the pyrroloindole structural motif in these natural compounds, a methyltransferase is needed for this key step in the biosynthetic pathway. , In nature, S -adenosyl methionine (SAM, 5 ) is used as a methyl donor for SAM-dependent methyltransferase reactions. The methyl group of SAM ( 5 ) can be transferred by these methyltransferases to a large variety of acceptor molecules, such as small metabolites or even biopolymers, whereby S -adenosyl-homocysteine (SAH) ( 6 ) is formed as the byproduct. − Methyltransferases can be further classified according to the atom on the substrate accepting the methyl group.…”