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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.


Related in: MedlinePlus

Enzymatic assay on the full-length NosA, NocA and their variants:(A) NosA and its K49A and E101A variants; (B) NosA and its E101D, E101Q, E101K and K49E variants; (C) NosA1-111, NosA1-111 mixed with NosA112-151 at mole ratio 1:1, and NosA112-151; (D) NocA and its K41A and E93A variants. In all cases, the substrate (top) and the product nosiheptide (bottom) were used as controls, highlighted in a dotted red line and green line, respectively.
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f3: Enzymatic assay on the full-length NosA, NocA and their variants:(A) NosA and its K49A and E101A variants; (B) NosA and its E101D, E101Q, E101K and K49E variants; (C) NosA1-111, NosA1-111 mixed with NosA112-151 at mole ratio 1:1, and NosA112-151; (D) NocA and its K41A and E93A variants. In all cases, the substrate (top) and the product nosiheptide (bottom) were used as controls, highlighted in a dotted red line and green line, respectively.

Mentions: To understand how NosA interacts with its substrate, we searched NosA structural homologs in protein data bank using DALI server9. The structures with Z-score higher than 7.0 were selected. They are heme-degrading enzymes101112131415 or HapK protein involved in prodigiosin biosynthesis16, demonstrating that their ligands bind to the region outside of the β-barrel (supporting information, Fig. S3). Based on these observations, we assumed that the substrate of NosA might also bind to the region out of the β-barrel. To confirm this, we performed alanine-scanning mutagenesis assay on the conserved residues that are located at the entrances of the β-barrel (such as C11 and Q48), within the β-barrel (such as R29 and S53), outside of the β-barrel (such as Q48, K49, W51 and E101), or in the C-terminal loop (such as F122, D123, P124, S126, D128, P129, R132, P133, E135, F136, P138 and P139) in the full-length NosA, respectively. Then, we measured the catalytic activities of these variants by running enzymatic assay on HPLC system (Fig. 3A and supporting information, Fig. S4). Only K49A and E101A variants abolish the catalytic activities, indicating that K49 and E101 might be key elements for catalytic reaction. As shown in Fig. 3B, when E101 was replaced by D101, Q101 or K101, and residue K49 was mutated into E49, respectively, the E101K, K49E and E101Q variants abolish or significantly lose catalytic activities, while E101D retains the catalytic activity, indicating that the charged side-chains of E101 and K49 are critical to the catalytic reaction.


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)

Enzymatic assay on the full-length NosA, NocA and their variants:(A) NosA and its K49A and E101A variants; (B) NosA and its E101D, E101Q, E101K and K49E variants; (C) NosA1-111, NosA1-111 mixed with NosA112-151 at mole ratio 1:1, and NosA112-151; (D) NocA and its K41A and E93A variants. In all cases, the substrate (top) and the product nosiheptide (bottom) were used as controls, highlighted in a dotted red line and green line, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Enzymatic assay on the full-length NosA, NocA and their variants:(A) NosA and its K49A and E101A variants; (B) NosA and its E101D, E101Q, E101K and K49E variants; (C) NosA1-111, NosA1-111 mixed with NosA112-151 at mole ratio 1:1, and NosA112-151; (D) NocA and its K41A and E93A variants. In all cases, the substrate (top) and the product nosiheptide (bottom) were used as controls, highlighted in a dotted red line and green line, respectively.
Mentions: To understand how NosA interacts with its substrate, we searched NosA structural homologs in protein data bank using DALI server9. The structures with Z-score higher than 7.0 were selected. They are heme-degrading enzymes101112131415 or HapK protein involved in prodigiosin biosynthesis16, demonstrating that their ligands bind to the region outside of the β-barrel (supporting information, Fig. S3). Based on these observations, we assumed that the substrate of NosA might also bind to the region out of the β-barrel. To confirm this, we performed alanine-scanning mutagenesis assay on the conserved residues that are located at the entrances of the β-barrel (such as C11 and Q48), within the β-barrel (such as R29 and S53), outside of the β-barrel (such as Q48, K49, W51 and E101), or in the C-terminal loop (such as F122, D123, P124, S126, D128, P129, R132, P133, E135, F136, P138 and P139) in the full-length NosA, respectively. Then, we measured the catalytic activities of these variants by running enzymatic assay on HPLC system (Fig. 3A and supporting information, Fig. S4). Only K49A and E101A variants abolish the catalytic activities, indicating that K49 and E101 might be key elements for catalytic reaction. As shown in Fig. 3B, when E101 was replaced by D101, Q101 or K101, and residue K49 was mutated into E49, respectively, the E101K, K49E and E101Q variants abolish or significantly lose catalytic activities, while E101D retains the catalytic activity, indicating that the charged side-chains of E101 and K49 are critical to the catalytic reaction.

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.


Related in: MedlinePlus