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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 coordinates with NosA112-151 to interact with the substrate.(A) 1H-15N HSQC spectra acquired on free NosA112-151, highlighted with NMR signal assignment of residues; (B) 1H-15N HSQC spectrum of NosA112-151 in complex with the substrate (in green) was overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; (C) 1H-15N HSQC spectrum of NosA112-151 in complex with NosA1-111 and substrate (in grey), overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; In all cases of (A–C), the concentration of NosA112-151 was about 0.2 mM in NMR buffer. (D) Relaxation time T1 measurements of backbone atoms of each residue in 15N-labeled NosA112-151 in its free state (black), in complex with NosA1-111 (green), in complex with the substrate (blue), in complex with NosA1-111 and substrate (red), respectively. (E) Two hydrogen bonds between NosA and the substrate are formed, supported by the distance changes during MD simulation trajectory. The change in the distance between nitrogen atom of the side-chain of K49 and oxygen atom in –OH group in pyridine of the substrate is shown in black, and the change in the distance between the backbone oxygen of S126 and oxygen atom in –OH group in the substrate highlighted in blue in Fig. 1 is shown in red, respectively. (F) The last snapshot of the MD simulation trajectory, where the two monomers of NosA were displayed in green and yellown ribbon modes, respectively. The substrate was displayed in cyan-sticks mode. The main residues contributing to the protein-substrate interactions, including K49 and E101 in NosA1-111 and residues in NosA1-120, were also shown in sticks mode. The two hydrogen-bonds between NosA and substrate were displayed in dotted lines.
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f5: NosA1-111 coordinates with NosA112-151 to interact with the substrate.(A) 1H-15N HSQC spectra acquired on free NosA112-151, highlighted with NMR signal assignment of residues; (B) 1H-15N HSQC spectrum of NosA112-151 in complex with the substrate (in green) was overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; (C) 1H-15N HSQC spectrum of NosA112-151 in complex with NosA1-111 and substrate (in grey), overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; In all cases of (A–C), the concentration of NosA112-151 was about 0.2 mM in NMR buffer. (D) Relaxation time T1 measurements of backbone atoms of each residue in 15N-labeled NosA112-151 in its free state (black), in complex with NosA1-111 (green), in complex with the substrate (blue), in complex with NosA1-111 and substrate (red), respectively. (E) Two hydrogen bonds between NosA and the substrate are formed, supported by the distance changes during MD simulation trajectory. The change in the distance between nitrogen atom of the side-chain of K49 and oxygen atom in –OH group in pyridine of the substrate is shown in black, and the change in the distance between the backbone oxygen of S126 and oxygen atom in –OH group in the substrate highlighted in blue in Fig. 1 is shown in red, respectively. (F) The last snapshot of the MD simulation trajectory, where the two monomers of NosA were displayed in green and yellown ribbon modes, respectively. The substrate was displayed in cyan-sticks mode. The main residues contributing to the protein-substrate interactions, including K49 and E101 in NosA1-111 and residues in NosA1-120, were also shown in sticks mode. The two hydrogen-bonds between NosA and substrate were displayed in dotted lines.

Mentions: To confirm this hypothesis, we performed the following biochemical assay. The CD spectrum of NosA112-151 reveals that the NosA112-151 peptide is disordered in its free state (supporting information, Fig. S7-A). Upon mixing with the N-terminal NosA1-111, the cross-peaks of the 1H-15N spectrum acquired on NosA112-151 are still not dispersed, mainly located in the region between 8.0 ppm and 8.5 ppm, similar to the observation in 1H-15N HSQC spectrum acquired on free NosA112-151 (Fig. 5A) (both spectra overlapped very well in supporting information, Fig. S7-B). This observation suggests that the C-terminal NosA112-151 peptide is still folded as a random coil conformer upon being mixed with NosA1-111. Moreover, the cross-peaks of the 1H-15N spectra acquired on NosA112-151 did not shift (supporting information, Fig. S7-B), indicating that NosA1-111 does interact very weakly with NosA112-151, consistent with the measurement of the binding affinity of NosA1-111 to NosA112-151 by ITC assay (supporting information, Fig. S6). Moreover, Titrating the substrate into 15N-labeled NosA112-151 solution only results in slight shift of several cross-peaks in 1H-15N HSQC of NosA112-151, suggesting that individual NosA112-151 interacts with the substrate weakly (Fig. 5B). Adding the substrate into the mixture of NosA1-111 and NosA112-151 leads to obvious, but still small chemical shift changes in the 1H-15N spectra (Fig. 5C), indicating that NosA1-111 may coordinate with NosA112-151 to interact with the substrate, consistent with the measurements of the kcat and Km values above. To further confirm this conclusion, we measured the dynamic properties of the backbone atoms (relaxation time T1 and T2 and 15N-1H NOE values) of free NosA112-151, and of NoxA112-151 mixed with NosA1-111, and of NoxA112-151 mixed with the substrate, and of NoxA112-151 mixed with both NosA1-111 and the substrate, respectively. The T1 values of the backbone atoms of NosA112-151 mixed with the substrate and the N-terminal NosA1-111 are the smallest among these cases (Fig. 5D), indicating that the conformation of the NosA112-151 peptide in the ternary complex is the most rigid among these cases. This observation reveals that the flexible conformation of NosA112-151 may be fixed in the presence of NosA1-111 and the substrate.


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 coordinates with NosA112-151 to interact with the substrate.(A) 1H-15N HSQC spectra acquired on free NosA112-151, highlighted with NMR signal assignment of residues; (B) 1H-15N HSQC spectrum of NosA112-151 in complex with the substrate (in green) was overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; (C) 1H-15N HSQC spectrum of NosA112-151 in complex with NosA1-111 and substrate (in grey), overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; In all cases of (A–C), the concentration of NosA112-151 was about 0.2 mM in NMR buffer. (D) Relaxation time T1 measurements of backbone atoms of each residue in 15N-labeled NosA112-151 in its free state (black), in complex with NosA1-111 (green), in complex with the substrate (blue), in complex with NosA1-111 and substrate (red), respectively. (E) Two hydrogen bonds between NosA and the substrate are formed, supported by the distance changes during MD simulation trajectory. The change in the distance between nitrogen atom of the side-chain of K49 and oxygen atom in –OH group in pyridine of the substrate is shown in black, and the change in the distance between the backbone oxygen of S126 and oxygen atom in –OH group in the substrate highlighted in blue in Fig. 1 is shown in red, respectively. (F) The last snapshot of the MD simulation trajectory, where the two monomers of NosA were displayed in green and yellown ribbon modes, respectively. The substrate was displayed in cyan-sticks mode. The main residues contributing to the protein-substrate interactions, including K49 and E101 in NosA1-111 and residues in NosA1-120, were also shown in sticks mode. The two hydrogen-bonds between NosA and substrate were displayed in dotted lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525488&req=5

f5: NosA1-111 coordinates with NosA112-151 to interact with the substrate.(A) 1H-15N HSQC spectra acquired on free NosA112-151, highlighted with NMR signal assignment of residues; (B) 1H-15N HSQC spectrum of NosA112-151 in complex with the substrate (in green) was overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; (C) 1H-15N HSQC spectrum of NosA112-151 in complex with NosA1-111 and substrate (in grey), overlapped with that of free NosA112-151 (in pink); the signals with chemical shifts changes were marked; In all cases of (A–C), the concentration of NosA112-151 was about 0.2 mM in NMR buffer. (D) Relaxation time T1 measurements of backbone atoms of each residue in 15N-labeled NosA112-151 in its free state (black), in complex with NosA1-111 (green), in complex with the substrate (blue), in complex with NosA1-111 and substrate (red), respectively. (E) Two hydrogen bonds between NosA and the substrate are formed, supported by the distance changes during MD simulation trajectory. The change in the distance between nitrogen atom of the side-chain of K49 and oxygen atom in –OH group in pyridine of the substrate is shown in black, and the change in the distance between the backbone oxygen of S126 and oxygen atom in –OH group in the substrate highlighted in blue in Fig. 1 is shown in red, respectively. (F) The last snapshot of the MD simulation trajectory, where the two monomers of NosA were displayed in green and yellown ribbon modes, respectively. The substrate was displayed in cyan-sticks mode. The main residues contributing to the protein-substrate interactions, including K49 and E101 in NosA1-111 and residues in NosA1-120, were also shown in sticks mode. The two hydrogen-bonds between NosA and substrate were displayed in dotted lines.
Mentions: To confirm this hypothesis, we performed the following biochemical assay. The CD spectrum of NosA112-151 reveals that the NosA112-151 peptide is disordered in its free state (supporting information, Fig. S7-A). Upon mixing with the N-terminal NosA1-111, the cross-peaks of the 1H-15N spectrum acquired on NosA112-151 are still not dispersed, mainly located in the region between 8.0 ppm and 8.5 ppm, similar to the observation in 1H-15N HSQC spectrum acquired on free NosA112-151 (Fig. 5A) (both spectra overlapped very well in supporting information, Fig. S7-B). This observation suggests that the C-terminal NosA112-151 peptide is still folded as a random coil conformer upon being mixed with NosA1-111. Moreover, the cross-peaks of the 1H-15N spectra acquired on NosA112-151 did not shift (supporting information, Fig. S7-B), indicating that NosA1-111 does interact very weakly with NosA112-151, consistent with the measurement of the binding affinity of NosA1-111 to NosA112-151 by ITC assay (supporting information, Fig. S6). Moreover, Titrating the substrate into 15N-labeled NosA112-151 solution only results in slight shift of several cross-peaks in 1H-15N HSQC of NosA112-151, suggesting that individual NosA112-151 interacts with the substrate weakly (Fig. 5B). Adding the substrate into the mixture of NosA1-111 and NosA112-151 leads to obvious, but still small chemical shift changes in the 1H-15N spectra (Fig. 5C), indicating that NosA1-111 may coordinate with NosA112-151 to interact with the substrate, consistent with the measurements of the kcat and Km values above. To further confirm this conclusion, we measured the dynamic properties of the backbone atoms (relaxation time T1 and T2 and 15N-1H NOE values) of free NosA112-151, and of NoxA112-151 mixed with NosA1-111, and of NoxA112-151 mixed with the substrate, and of NoxA112-151 mixed with both NosA1-111 and the substrate, respectively. The T1 values of the backbone atoms of NosA112-151 mixed with the substrate and the N-terminal NosA1-111 are the smallest among these cases (Fig. 5D), indicating that the conformation of the NosA112-151 peptide in the ternary complex is the most rigid among these cases. This observation reveals that the flexible conformation of NosA112-151 may be fixed in the presence of NosA1-111 and the substrate.

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.