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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.


The folding and aggregation states of full-length NosA, NosA1-111 and its variants.(A–D) The aggregation states detected by size-exclusion chromatography assay; In (D), gel filtration protein standard markers were highlighted with arabic numerals 1, 2, 3 and 4, representing thyroglobulin with a molecular weight (MW) of 670KDa, γ-globulin with a MW of 158 KDa, Ovalbumin with a MW of 44 KDa, myoglobin with MW of 17 KDa and vitamin B12 with a MW of 1.35 KDa, respectively. (E) The folding of NosA and its variants detected by circular dichroism (CD) spectroscopies respectively; (F) The folding of NosA K49A variant further confirmed by two-dimensional NMR 1H-15N HSQC spectrum of the full-length NosA K49A variant (green), overlapped with that of wild-type NosA (red). In these two NMR experiments, the concentration of the WT NosA protein and its K49A variant is about 0.2 mM in NMR buffer.
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f4: The folding and aggregation states of full-length NosA, NosA1-111 and its variants.(A–D) The aggregation states detected by size-exclusion chromatography assay; In (D), gel filtration protein standard markers were highlighted with arabic numerals 1, 2, 3 and 4, representing thyroglobulin with a molecular weight (MW) of 670KDa, γ-globulin with a MW of 158 KDa, Ovalbumin with a MW of 44 KDa, myoglobin with MW of 17 KDa and vitamin B12 with a MW of 1.35 KDa, respectively. (E) The folding of NosA and its variants detected by circular dichroism (CD) spectroscopies respectively; (F) The folding of NosA K49A variant further confirmed by two-dimensional NMR 1H-15N HSQC spectrum of the full-length NosA K49A variant (green), overlapped with that of wild-type NosA (red). In these two NMR experiments, the concentration of the WT NosA protein and its K49A variant is about 0.2 mM in NMR buffer.

Mentions: Finally, we tested the possible effects on the folding and the aggregation state of NosA by these mutations by running the size-exclusion chromatography (SEC) assay, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The results from the SEC assay suggested that the aggregation states of these variants were not affected by these mutations (Fig. 4A–C). Moreover, as shown in Fig. 4D, the retention time of the WT full-length NosA and the truncated NosA1-111 locates between gel filtration protein markers (3 and 4) with molecular weights of 44KDa and 17KDa, respectively, indicating that they are dimer in solution. Thus, the C-terminal loop is not helpful to NosA dimerization. However, the 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. Thus, the loss of the catalytic activity of K49A might also result from the changes in the folding of the protein.


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 folding and aggregation states of full-length NosA, NosA1-111 and its variants.(A–D) The aggregation states detected by size-exclusion chromatography assay; In (D), gel filtration protein standard markers were highlighted with arabic numerals 1, 2, 3 and 4, representing thyroglobulin with a molecular weight (MW) of 670KDa, γ-globulin with a MW of 158 KDa, Ovalbumin with a MW of 44 KDa, myoglobin with MW of 17 KDa and vitamin B12 with a MW of 1.35 KDa, respectively. (E) The folding of NosA and its variants detected by circular dichroism (CD) spectroscopies respectively; (F) The folding of NosA K49A variant further confirmed by two-dimensional NMR 1H-15N HSQC spectrum of the full-length NosA K49A variant (green), overlapped with that of wild-type NosA (red). In these two NMR experiments, the concentration of the WT NosA protein and its K49A variant is about 0.2 mM in NMR buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The folding and aggregation states of full-length NosA, NosA1-111 and its variants.(A–D) The aggregation states detected by size-exclusion chromatography assay; In (D), gel filtration protein standard markers were highlighted with arabic numerals 1, 2, 3 and 4, representing thyroglobulin with a molecular weight (MW) of 670KDa, γ-globulin with a MW of 158 KDa, Ovalbumin with a MW of 44 KDa, myoglobin with MW of 17 KDa and vitamin B12 with a MW of 1.35 KDa, respectively. (E) The folding of NosA and its variants detected by circular dichroism (CD) spectroscopies respectively; (F) The folding of NosA K49A variant further confirmed by two-dimensional NMR 1H-15N HSQC spectrum of the full-length NosA K49A variant (green), overlapped with that of wild-type NosA (red). In these two NMR experiments, the concentration of the WT NosA protein and its K49A variant is about 0.2 mM in NMR buffer.
Mentions: Finally, we tested the possible effects on the folding and the aggregation state of NosA by these mutations by running the size-exclusion chromatography (SEC) assay, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The results from the SEC assay suggested that the aggregation states of these variants were not affected by these mutations (Fig. 4A–C). Moreover, as shown in Fig. 4D, the retention time of the WT full-length NosA and the truncated NosA1-111 locates between gel filtration protein markers (3 and 4) with molecular weights of 44KDa and 17KDa, respectively, indicating that they are dimer in solution. Thus, the C-terminal loop is not helpful to NosA dimerization. However, the 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. Thus, the loss of the catalytic activity of K49A might also result from the changes in the folding of the protein.

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.