Biosynthesis of the nosiheptide indole side ring centers on a cryptic carrier protein NosJ

Abstract

Nosiheptide is a prototypal thiopeptide antibiotic, containing an indole side ring in addition to its thiopeptide-characteristic macrocylic scaffold. This indole ring is derived from 3-methyl-2-indolic acid (MIA), a product of the radical S-adenosylmethionine enzyme NosL, but how MIA is incorporated into nosiheptide biosynthesis remains to be investigated. Here we report functional dissection of a series of enzymes involved in nosiheptide biosynthesis. We show NosI activates MIA and transfers it to the phosphopantetheinyl arm of a carrier protein NosJ. NosN then acts on the NosJ-bound MIA and installs a methyl group on the indole C4, and the resulting dimethylindolyl moiety is released from NosJ by a hydrolase-like enzyme NosK. Surface plasmon resonance analysis show that the molecular complex of NosJ with NosN is much more stable than those with other enzymes, revealing an elegant biosynthetic strategy in which the reaction flux is controlled by protein–protein interactions with different binding affinities.

NosN, a Radical S-Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide

“…More recently, the Zhang laboratory also showed that NosJ-MIA also serves as a substrate for NosN, which it converts to NosJ-DMIA. 6 …”

The hidden enzymology of bacterial natural product biosynthesis

“…Unusually, NosL initiates this intricate transformation by abstracting an H-atom from the α-amino group, rather than a carbon centre [148][149][150][151] . The subsequent formation of the indolic side-ring system from MIA involves the action of a novel, discrete, indoyl thiolation protein that was previously unannotated (NosJ) and a unique α/β-hydrolase fold protein (NosK) that transfers the indoyl to a linear pentathiazolyl peptide intermediate Cys residue that remains selectively unmodified [152][153][154] (FIG. 9).…”

Using Peptide Mimics to Study the Biosynthesis of the Side-Ring System of Nosiheptide

“…NosN has been stated to produce 5′-deoxyadenosine (5′-dA) and either S-adenosylhomocysteine (SAH) or thioadenosine (tA), depending on whether S-adenosylmethionine (SAM) or methylthioadenosine (MTA) acts as a methyl donor, respectively (Ding, Li, et al, 2017; LaMattina et al, 2017). SAM typically degrades to MTA during long periods of storage, which is heavily dependent on the pH of the storage solution and the temperature.…”

“…Biosynthesis of the side-ring begins with NosL, which catalyzes the C α –C β fragmentation–recombination of L-Trp to afford MIA (Bhandari, Fedoseyenko, & Begley, 2016; Sicoli et al, 2016; Zhang et al, 2011). AMP is then attached to the carboxyl group of MIA by NosI to allow for transfer of MIA to the 4′-phosphopantetheine prosthetic group on NosJ (Badding et al, 2017; Ding, Ji, et al, 2017; Qiu et al, 2017). NosJ then shuttles MIA to NosK, an α/β hydrolase that attaches MIA onto a seryl residue within its active site.…”