Structure-based Mechanistic Insights into Terminal Amide Synthase in Nosiheptide-Represented Thiopeptides Biosynthesis.

Shanshan Liu,Wenxian Lan,Li Han,Wen Liu,Heng Guo,Naiyan Rong,Jianping Ding,Yan Zhang,Tianlong Zhang,Yi Yu,Renxiao Wang,Chunxi Wang,Chunyang Cao,Pengfei Yao

doi:10.1038/srep12744

Abstract

Nosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

Highlights

The results from the Size-exclusion chromatography (SEC) assay suggested that the aggregation states of these variants were not affected by these mutations (Fig. 4A–C)
The circular dichroism (CD) spectrum of the K49A variant looks much different from those of WT protein and other variants (Fig. 4E), and its 2D 1H-15N HSQC spectrum did not overlap well with that of WT NosA (Fig. 4F), indicating that the mutation from K49 to A49 might affect the folding of the protein
The loss of the catalytic activity of K49A might result from the changes in the folding of the protein

Summary

Synthase in NosiheptideRepresented Thiopeptides Biosynthesis

Shanshan Liu[1], Heng Guo1,*, Tianlong Zhang2,*, Li Han1,*, Pengfei Yao1,*, Yan Zhang[1], Naiyan Rong[1], Yi Yu1, Wenxian Lan[1], Chunxi Wang[1], Jianping Ding[2], Renxiao Wang[1], Wen Liu1 & Chunyang Cao[1]. Thiopeptides are a class of sulfur-rich, highly modified peptide antibiotics that are active against various drug-resistant bacterial pathogens These antibiotics share a common ribosomally synthesized paradigm in biosynthesis, featuring conserved post-translational modifications of a precursor peptide to afford a family-characteristic framework in which a nitrogen-containing, six-membered ring is central to multiple azoles and dehydroamino acids. The amino group of the terminal amide moiety in nosiheptide is endogenous and derives from an extended Ser residue of the precursor peptide[3,4]. Dehydration of this residue at the early stage in the nosiheptide biosynthetic pathway. We provide the structural basis of NosA for investigation into its enzymatic mechanism

Results and Discussion

Methods

Author Contributions

Additional Information