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Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis.

Sauguet L, Moutiez M, Li Y, Belin P, Seguin J, Le Du MH, Thai R, Masson C, Fonvielle M, Pernodet JL, Charbonnier JB, Gondry M - Nucleic Acids Res. (2011)

Bottom Line: These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs.AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine.These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

View Article: PubMed Central - PubMed

Affiliation: CEA, IBITECS, Service d'Ingénierie Moléculaire des Protéines, F-91191 Gif-sur-Yvette, France.

ABSTRACT
Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

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Identification of the covalent aminoacyl-enzyme intermediate. (A) Peptide mass fingerprint (PMF) analysis of trypsin-digested AlbC pre-incubated with Phe-tRNAPhe. Arrows indicate the formation of two additional fragments compared to PMF analysis of trypsin-digested AlbC not incubated with Phe-tRNAPhe (Supplementary Figure S7): one at m/z 2026.00 (continuous line frame) corresponding to phenylalanylated fragment [29–46] at m/z 1878.94, and the other at m/z 2310.17 (dashed line frame) corresponding to phenylalanylated fragment [27–46] at m/z 2163.08. (B) PSD-MS/MS spectrum of m/z 2026.00. Both b- and y-ions series identify the amino acids sequence of the modified AlbC fragment [29–46], and the residue 37 as the phenylalanylated residue. PSD-MS/MS spectrum of m/z 2310.17 is shown in Supplementary Figure S8.
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Figure 7: Identification of the covalent aminoacyl-enzyme intermediate. (A) Peptide mass fingerprint (PMF) analysis of trypsin-digested AlbC pre-incubated with Phe-tRNAPhe. Arrows indicate the formation of two additional fragments compared to PMF analysis of trypsin-digested AlbC not incubated with Phe-tRNAPhe (Supplementary Figure S7): one at m/z 2026.00 (continuous line frame) corresponding to phenylalanylated fragment [29–46] at m/z 1878.94, and the other at m/z 2310.17 (dashed line frame) corresponding to phenylalanylated fragment [27–46] at m/z 2163.08. (B) PSD-MS/MS spectrum of m/z 2026.00. Both b- and y-ions series identify the amino acids sequence of the modified AlbC fragment [29–46], and the residue 37 as the phenylalanylated residue. PSD-MS/MS spectrum of m/z 2310.17 is shown in Supplementary Figure S8.

Mentions: In order to unambiguously identify the residue acylated and the nature of the acylation, we performed peptide mass fingerprint (PMF) analyses on trypsin-digested AlbC, previously incubated or not with Phe-tRNAPhe. AlbC without substrate incubation gave two identified fragments containing the residue S37 (fragments [29–46] and [27–46]) (Supplementary Figure S7A). The corresponding m/z of these fragments (calculated m/z 1878.94 and 2163.09, respectively) were selected and isolated as precursor ion for PSD MS/MS sequence characterization (Supplementary Figure S7B and C). The same experiment performed on AlbC incubated with Phe-tRNAPhe gave two additional fragments (Figure 7A). The m/z of these two fragments were 2026.01 (observed m/z 2026.00) and 2310.16 (observed m/z 2310.17), and corresponded to the values expected for the addition of a phenylalanyl moiety (addition of 147.07) on fragments [29–46] and [27–46], respectively. These fragments, sequenced unambiguously by both b- and y-ions series, contained a phenylalanyl moiety on S37 (Figure 7B and Supplementary Figure S8). This demonstrated the formation of a covalent intermediate, in which l-Phe is transferred from Phe-tRNAPhe to S37 during the catalytic cycle. Moreover, the same experiment was performed on relevant AlbC variants. No modified fragments were observed for the variant S37A (Supplementary Figure S9). For the variants E182Q and Y178F, phenylalanylated fragments could be significantly detected, but in much less amounts than those obtained with the wild-type enzyme (Supplementary Figures S10 and S11), in agreement with radiolabelling experiments (Figure 6). These fragments were formed with amounts insufficient for PSD-MS/MS analyses (Supplementary Figures S10 and S11). For the variant Y202F, the phenylalanylated fragments were observed in proportions similar to those obtained with the wild-type enzyme, and were unambiguously sequenced (Supplementary Figure S12). The results confirm that Y202 is not responsible for S37 activation. For the variant S37C, the phenylalanylation of the fragments [27–49] and [29–46] (respectively, calculated m/z of 2040.97 and 2326.13 instead of 1894.90 and 2179.06) was detected (Supplementary Figure S13A). MS/MS sequencing of both m/z demonstrated the formation of a thioester link between the phenylalanyl moiety and the thiol group of C37 (Supplementary Figure S13B and C). All these data allow the proposal of a mechanism for the formation of a covalent intermediate during the catalytic cycle of the CDPS AlbC (see Discussion section).Figure 7.


Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis.

Sauguet L, Moutiez M, Li Y, Belin P, Seguin J, Le Du MH, Thai R, Masson C, Fonvielle M, Pernodet JL, Charbonnier JB, Gondry M - Nucleic Acids Res. (2011)

Identification of the covalent aminoacyl-enzyme intermediate. (A) Peptide mass fingerprint (PMF) analysis of trypsin-digested AlbC pre-incubated with Phe-tRNAPhe. Arrows indicate the formation of two additional fragments compared to PMF analysis of trypsin-digested AlbC not incubated with Phe-tRNAPhe (Supplementary Figure S7): one at m/z 2026.00 (continuous line frame) corresponding to phenylalanylated fragment [29–46] at m/z 1878.94, and the other at m/z 2310.17 (dashed line frame) corresponding to phenylalanylated fragment [27–46] at m/z 2163.08. (B) PSD-MS/MS spectrum of m/z 2026.00. Both b- and y-ions series identify the amino acids sequence of the modified AlbC fragment [29–46], and the residue 37 as the phenylalanylated residue. PSD-MS/MS spectrum of m/z 2310.17 is shown in Supplementary Figure S8.
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Figure 7: Identification of the covalent aminoacyl-enzyme intermediate. (A) Peptide mass fingerprint (PMF) analysis of trypsin-digested AlbC pre-incubated with Phe-tRNAPhe. Arrows indicate the formation of two additional fragments compared to PMF analysis of trypsin-digested AlbC not incubated with Phe-tRNAPhe (Supplementary Figure S7): one at m/z 2026.00 (continuous line frame) corresponding to phenylalanylated fragment [29–46] at m/z 1878.94, and the other at m/z 2310.17 (dashed line frame) corresponding to phenylalanylated fragment [27–46] at m/z 2163.08. (B) PSD-MS/MS spectrum of m/z 2026.00. Both b- and y-ions series identify the amino acids sequence of the modified AlbC fragment [29–46], and the residue 37 as the phenylalanylated residue. PSD-MS/MS spectrum of m/z 2310.17 is shown in Supplementary Figure S8.
Mentions: In order to unambiguously identify the residue acylated and the nature of the acylation, we performed peptide mass fingerprint (PMF) analyses on trypsin-digested AlbC, previously incubated or not with Phe-tRNAPhe. AlbC without substrate incubation gave two identified fragments containing the residue S37 (fragments [29–46] and [27–46]) (Supplementary Figure S7A). The corresponding m/z of these fragments (calculated m/z 1878.94 and 2163.09, respectively) were selected and isolated as precursor ion for PSD MS/MS sequence characterization (Supplementary Figure S7B and C). The same experiment performed on AlbC incubated with Phe-tRNAPhe gave two additional fragments (Figure 7A). The m/z of these two fragments were 2026.01 (observed m/z 2026.00) and 2310.16 (observed m/z 2310.17), and corresponded to the values expected for the addition of a phenylalanyl moiety (addition of 147.07) on fragments [29–46] and [27–46], respectively. These fragments, sequenced unambiguously by both b- and y-ions series, contained a phenylalanyl moiety on S37 (Figure 7B and Supplementary Figure S8). This demonstrated the formation of a covalent intermediate, in which l-Phe is transferred from Phe-tRNAPhe to S37 during the catalytic cycle. Moreover, the same experiment was performed on relevant AlbC variants. No modified fragments were observed for the variant S37A (Supplementary Figure S9). For the variants E182Q and Y178F, phenylalanylated fragments could be significantly detected, but in much less amounts than those obtained with the wild-type enzyme (Supplementary Figures S10 and S11), in agreement with radiolabelling experiments (Figure 6). These fragments were formed with amounts insufficient for PSD-MS/MS analyses (Supplementary Figures S10 and S11). For the variant Y202F, the phenylalanylated fragments were observed in proportions similar to those obtained with the wild-type enzyme, and were unambiguously sequenced (Supplementary Figure S12). The results confirm that Y202 is not responsible for S37 activation. For the variant S37C, the phenylalanylation of the fragments [27–49] and [29–46] (respectively, calculated m/z of 2040.97 and 2326.13 instead of 1894.90 and 2179.06) was detected (Supplementary Figure S13A). MS/MS sequencing of both m/z demonstrated the formation of a thioester link between the phenylalanyl moiety and the thiol group of C37 (Supplementary Figure S13B and C). All these data allow the proposal of a mechanism for the formation of a covalent intermediate during the catalytic cycle of the CDPS AlbC (see Discussion section).Figure 7.

Bottom Line: These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs.AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine.These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

View Article: PubMed Central - PubMed

Affiliation: CEA, IBITECS, Service d'Ingénierie Moléculaire des Protéines, F-91191 Gif-sur-Yvette, France.

ABSTRACT
Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

Show MeSH