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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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Crystal structure of S. noursei AlbC and comparison with that of M. tuberculosis Rv2275. (A) Overall structure of AlbC (region 28–236) in cartoon mode, with α helices, β-strands, and loops coloured in green, yellow and grey, respectively. AlbC secondary-structure elements are numbered according to Rv2275 numbering (12). The Rossmann-fold domain is constituted by helix α2, the C-terminal part of helix 4 and the β-sheet (β3–β5) that is extended by two additional strands β6 and β7. A helical region (in light green), composed of helices α5, α6 and α7, packs against the Rossmann fold. (B) Structure of the AlbC surface-accessible pocket. Residues that delineate the AlbC pocket are shown in ball and stick representation. The most buried part of the pocket is mainly constituted of hydrophobic residues (coloured in green). The entrance of the pocket is mainly delineated by polar residues (coloured in orange). The boundaries of the pocket, calculated using the program CAVER (58), are defined by a semi-transparent grey surface. The molecules of phosphate and cDTT modelled in the electron density are shown in orange and dark green, respectively. (C) Superimposition of the structures of AlbC and Rv2275 in cartoon representation. AlbC and Rv2275 are coloured in green and magenta, respectively. Both proteins superpose well. Most significant structural differences concern N- and C-terminus regions, and also relative positions of helices α5, α6 and α8. (D) Superimposition of the surface-accessible pockets of AlbC and Rv2275, with an orientation similar to that shown in Figure 2C. Residues represented in ball and stick are essential for CDPS activity. AlbC and Rv2275 residues are indicated by A and R letters, respectively. The superimposition highlights the structural deviations between loops β3–α2 and β6–α2, and residues Y202A and Y253R located at the entrance of the active sites.
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Figure 2: Crystal structure of S. noursei AlbC and comparison with that of M. tuberculosis Rv2275. (A) Overall structure of AlbC (region 28–236) in cartoon mode, with α helices, β-strands, and loops coloured in green, yellow and grey, respectively. AlbC secondary-structure elements are numbered according to Rv2275 numbering (12). The Rossmann-fold domain is constituted by helix α2, the C-terminal part of helix 4 and the β-sheet (β3–β5) that is extended by two additional strands β6 and β7. A helical region (in light green), composed of helices α5, α6 and α7, packs against the Rossmann fold. (B) Structure of the AlbC surface-accessible pocket. Residues that delineate the AlbC pocket are shown in ball and stick representation. The most buried part of the pocket is mainly constituted of hydrophobic residues (coloured in green). The entrance of the pocket is mainly delineated by polar residues (coloured in orange). The boundaries of the pocket, calculated using the program CAVER (58), are defined by a semi-transparent grey surface. The molecules of phosphate and cDTT modelled in the electron density are shown in orange and dark green, respectively. (C) Superimposition of the structures of AlbC and Rv2275 in cartoon representation. AlbC and Rv2275 are coloured in green and magenta, respectively. Both proteins superpose well. Most significant structural differences concern N- and C-terminus regions, and also relative positions of helices α5, α6 and α8. (D) Superimposition of the surface-accessible pockets of AlbC and Rv2275, with an orientation similar to that shown in Figure 2C. Residues represented in ball and stick are essential for CDPS activity. AlbC and Rv2275 residues are indicated by A and R letters, respectively. The superimposition highlights the structural deviations between loops β3–α2 and β6–α2, and residues Y202A and Y253R located at the entrance of the active sites.

Mentions: We produced AlbC in E. coli, purified it to homogeneity (1) and crystallized the purified AlbC. We solved the crystal structure of AlbC by the multi-wavelength anomalous dispersion (MAD) method using selenomethionine-labelled protein. The native structure was refined to a final R-factor/R-free of 18.6/23.6% at a resolution of 1.9 Å (Table 1). The crystal structure of AlbC is a monomer that presents a compact α/β structure (Figure 2). It is composed of a central β-sheet with five parallel β strands (β3–β7) surrounded by ten α helices (α2–α9) (Figure 2A). The AlbC structure contains a Rossmann-fold domain (27) formed by strands β3–β5, helix α2 and the C-terminal part of helix α4. This domain is followed by a helical region composed of three main helices (α5–α7) that pack against the Rossmann fold. The structure ends with two β strands (β6 and β7) that extend the Rossmann β-sheet and two small connecting helices (α8 and α9). AlbC residues 1–27 were not visible in the electron density map. Crystal packing analysis showed the presence of large cavities near the residue 28 that are compatible with the presence of a flexible N-terminal extension (Supplementary Figure S2). Some regions of AlbC were visible in the electron density map, but they present higher B-factors than the rest of the protein (Supplementary Figure S3). These regions contain the residues 38–46 in loop β3–α2, the residues 105–111 in loop α4–β5, the residues 215–220 in loop α8–β7, and the C-terminal residues 236–239.Figure 2.


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)

Crystal structure of S. noursei AlbC and comparison with that of M. tuberculosis Rv2275. (A) Overall structure of AlbC (region 28–236) in cartoon mode, with α helices, β-strands, and loops coloured in green, yellow and grey, respectively. AlbC secondary-structure elements are numbered according to Rv2275 numbering (12). The Rossmann-fold domain is constituted by helix α2, the C-terminal part of helix 4 and the β-sheet (β3–β5) that is extended by two additional strands β6 and β7. A helical region (in light green), composed of helices α5, α6 and α7, packs against the Rossmann fold. (B) Structure of the AlbC surface-accessible pocket. Residues that delineate the AlbC pocket are shown in ball and stick representation. The most buried part of the pocket is mainly constituted of hydrophobic residues (coloured in green). The entrance of the pocket is mainly delineated by polar residues (coloured in orange). The boundaries of the pocket, calculated using the program CAVER (58), are defined by a semi-transparent grey surface. The molecules of phosphate and cDTT modelled in the electron density are shown in orange and dark green, respectively. (C) Superimposition of the structures of AlbC and Rv2275 in cartoon representation. AlbC and Rv2275 are coloured in green and magenta, respectively. Both proteins superpose well. Most significant structural differences concern N- and C-terminus regions, and also relative positions of helices α5, α6 and α8. (D) Superimposition of the surface-accessible pockets of AlbC and Rv2275, with an orientation similar to that shown in Figure 2C. Residues represented in ball and stick are essential for CDPS activity. AlbC and Rv2275 residues are indicated by A and R letters, respectively. The superimposition highlights the structural deviations between loops β3–α2 and β6–α2, and residues Y202A and Y253R located at the entrance of the active sites.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Crystal structure of S. noursei AlbC and comparison with that of M. tuberculosis Rv2275. (A) Overall structure of AlbC (region 28–236) in cartoon mode, with α helices, β-strands, and loops coloured in green, yellow and grey, respectively. AlbC secondary-structure elements are numbered according to Rv2275 numbering (12). The Rossmann-fold domain is constituted by helix α2, the C-terminal part of helix 4 and the β-sheet (β3–β5) that is extended by two additional strands β6 and β7. A helical region (in light green), composed of helices α5, α6 and α7, packs against the Rossmann fold. (B) Structure of the AlbC surface-accessible pocket. Residues that delineate the AlbC pocket are shown in ball and stick representation. The most buried part of the pocket is mainly constituted of hydrophobic residues (coloured in green). The entrance of the pocket is mainly delineated by polar residues (coloured in orange). The boundaries of the pocket, calculated using the program CAVER (58), are defined by a semi-transparent grey surface. The molecules of phosphate and cDTT modelled in the electron density are shown in orange and dark green, respectively. (C) Superimposition of the structures of AlbC and Rv2275 in cartoon representation. AlbC and Rv2275 are coloured in green and magenta, respectively. Both proteins superpose well. Most significant structural differences concern N- and C-terminus regions, and also relative positions of helices α5, α6 and α8. (D) Superimposition of the surface-accessible pockets of AlbC and Rv2275, with an orientation similar to that shown in Figure 2C. Residues represented in ball and stick are essential for CDPS activity. AlbC and Rv2275 residues are indicated by A and R letters, respectively. The superimposition highlights the structural deviations between loops β3–α2 and β6–α2, and residues Y202A and Y253R located at the entrance of the active sites.
Mentions: We produced AlbC in E. coli, purified it to homogeneity (1) and crystallized the purified AlbC. We solved the crystal structure of AlbC by the multi-wavelength anomalous dispersion (MAD) method using selenomethionine-labelled protein. The native structure was refined to a final R-factor/R-free of 18.6/23.6% at a resolution of 1.9 Å (Table 1). The crystal structure of AlbC is a monomer that presents a compact α/β structure (Figure 2). It is composed of a central β-sheet with five parallel β strands (β3–β7) surrounded by ten α helices (α2–α9) (Figure 2A). The AlbC structure contains a Rossmann-fold domain (27) formed by strands β3–β5, helix α2 and the C-terminal part of helix α4. This domain is followed by a helical region composed of three main helices (α5–α7) that pack against the Rossmann fold. The structure ends with two β strands (β6 and β7) that extend the Rossmann β-sheet and two small connecting helices (α8 and α9). AlbC residues 1–27 were not visible in the electron density map. Crystal packing analysis showed the presence of large cavities near the residue 28 that are compatible with the presence of a flexible N-terminal extension (Supplementary Figure S2). Some regions of AlbC were visible in the electron density map, but they present higher B-factors than the rest of the protein (Supplementary Figure S3). These regions contain the residues 38–46 in loop β3–α2, the residues 105–111 in loop α4–β5, the residues 215–220 in loop α8–β7, and the C-terminal residues 236–239.Figure 2.

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
Related in: MedlinePlus