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.


NosA1-111 overall fold.(A): vertical view, (B): lateral view) Ribbon representations of a NosA trimer observed in an asymmetric unit, monomers were highlighted in red, blue and yellow, respectively. (C) monomer conformation, N-terminal and C-terminal and secondary structures were marked; (D) residues forming the hydrogen bonds in the β4 strand and the β2’ strand outside of β-barrel; (E) the salt-bridge and hydrogen-bonds formation between R96 and E94’ within the β-barrel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: NosA1-111 overall fold.(A): vertical view, (B): lateral view) Ribbon representations of a NosA trimer observed in an asymmetric unit, monomers were highlighted in red, blue and yellow, respectively. (C) monomer conformation, N-terminal and C-terminal and secondary structures were marked; (D) residues forming the hydrogen bonds in the β4 strand and the β2’ strand outside of β-barrel; (E) the salt-bridge and hydrogen-bonds formation between R96 and E94’ within the β-barrel.

Mentions: Three monomers occupy one asymmetric unit (Fig. 2A,B), two of them form a dimer. One monomer constitutes a dimer with one monomer in an adjacent asymmetric unit. Each monomer is identical to the others with an RMSD value of 0.21 Å for the backbone Cα atoms in the secondary structural regions, consisting of four anti-parallel β-sheets (β1, β2, β3, and β4), three α-helices (α1, α2 and α3) and six loops (L1, L2, L3, L4, L5 and L6), which are arranged in the order of β1- L1- α1- L2- β2- L3- β3- L4- α2- L5- α3- L6- β4 (Fig. 2C). The anti-parallel β-sheets form a semicircular hydrophobic surface, the α-helices and loops are located on the outside of the circle. The first six or fewer residues at the N-terminus are invisible in all monomers, and the residues from 35 to 42 are also invisible in the two monomers that form a dimer conformation. This dimeric structure adopts a global fold, resembling an elliptic β-barrel of 28.9 Å in height, with diameters of 15 Å and 23 Å (Fig. 2A). The α-helices and loops surround the β-barrel.


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)

NosA1-111 overall fold.(A): vertical view, (B): lateral view) Ribbon representations of a NosA trimer observed in an asymmetric unit, monomers were highlighted in red, blue and yellow, respectively. (C) monomer conformation, N-terminal and C-terminal and secondary structures were marked; (D) residues forming the hydrogen bonds in the β4 strand and the β2’ strand outside of β-barrel; (E) the salt-bridge and hydrogen-bonds formation between R96 and E94’ within the β-barrel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: NosA1-111 overall fold.(A): vertical view, (B): lateral view) Ribbon representations of a NosA trimer observed in an asymmetric unit, monomers were highlighted in red, blue and yellow, respectively. (C) monomer conformation, N-terminal and C-terminal and secondary structures were marked; (D) residues forming the hydrogen bonds in the β4 strand and the β2’ strand outside of β-barrel; (E) the salt-bridge and hydrogen-bonds formation between R96 and E94’ within the β-barrel.
Mentions: Three monomers occupy one asymmetric unit (Fig. 2A,B), two of them form a dimer. One monomer constitutes a dimer with one monomer in an adjacent asymmetric unit. Each monomer is identical to the others with an RMSD value of 0.21 Å for the backbone Cα atoms in the secondary structural regions, consisting of four anti-parallel β-sheets (β1, β2, β3, and β4), three α-helices (α1, α2 and α3) and six loops (L1, L2, L3, L4, L5 and L6), which are arranged in the order of β1- L1- α1- L2- β2- L3- β3- L4- α2- L5- α3- L6- β4 (Fig. 2C). The anti-parallel β-sheets form a semicircular hydrophobic surface, the α-helices and loops are located on the outside of the circle. The first six or fewer residues at the N-terminus are invisible in all monomers, and the residues from 35 to 42 are also invisible in the two monomers that form a dimer conformation. This dimeric structure adopts a global fold, resembling an elliptic β-barrel of 28.9 Å in height, with diameters of 15 Å and 23 Å (Fig. 2A). The α-helices and loops surround the β-barrel.

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.