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

Liu S, Guo H, Zhang T, Han L, Yao P, Zhang Y, Rong N, Yu Y, Lan W, Wang C, Ding J, Wang R, Liu W, Cao C - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bio-Organic and Natural Product Chemistry and Collaborative Innovation Center of Chemistry for Life Sciences, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.

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.

No MeSH data available.


The proposed mechanism of NosA to catalyze nosiheptide maturation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525488&req=5

f7: The proposed mechanism of NosA to catalyze nosiheptide maturation.

Mentions: Taken all results above together, we proposed the following catalytic mechanism (Fig. 7): (1) The terminal dehydroalanine unit is tautomerized to methyl imine intermediate A in basic buffer condition, supported by the previous studies on thiostrepton synthesis2324, where similar reaction is initiated by Et2NH; (2) The substrate is fixed into the active sites by hydrogen-bond and hydrophobic interactions between the substrate and residue K49, the C-terminus of NosA, generating intermediate B, supported by the structural and MD studies above; (3) The negatively charged side-chain of E101’ interacts with one molecule H2O, supported by the findings that several water molecules exist close to E101’ in the crystal structure; (4) The nucleophilic attack by H2O to methyl imine produces intermediates C and D, leading to the final Cα-N bond cleavage to yield nosiheptide and pyruvate.


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

Liu S, Guo H, Zhang T, Han L, Yao P, Zhang Y, Rong N, Yu Y, Lan W, Wang C, Ding J, Wang R, Liu W, Cao C - Sci Rep (2015)

The proposed mechanism of NosA to catalyze nosiheptide maturation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525488&req=5

f7: The proposed mechanism of NosA to catalyze nosiheptide maturation.
Mentions: Taken all results above together, we proposed the following catalytic mechanism (Fig. 7): (1) The terminal dehydroalanine unit is tautomerized to methyl imine intermediate A in basic buffer condition, supported by the previous studies on thiostrepton synthesis2324, where similar reaction is initiated by Et2NH; (2) The substrate is fixed into the active sites by hydrogen-bond and hydrophobic interactions between the substrate and residue K49, the C-terminus of NosA, generating intermediate B, supported by the structural and MD studies above; (3) The negatively charged side-chain of E101’ interacts with one molecule H2O, supported by the findings that several water molecules exist close to E101’ in the crystal structure; (4) The nucleophilic attack by H2O to methyl imine produces intermediates C and D, leading to the final Cα-N bond cleavage to yield nosiheptide and pyruvate.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bio-Organic and Natural Product Chemistry and Collaborative Innovation Center of Chemistry for Life Sciences, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.

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.

No MeSH data available.