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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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Superimposition of the structures of AlbC and TyrRSMj in complex with its l-tyrosine substrate (PDB id: 1j1u). The two enzymes are shown in cartoon mode, and l-tyrosine is in ball and stick coloured in orange. The Rossmann-fold and the CP1 domains of TyrRSMj are coloured in dark and light blue, respectively, and the corresponding domains of AlbC are coloured in dark and light green, respectively. The Rossmann-fold and CP1 domains of the two proteins have similar rmsd values (3.25 Å over 96 Cα and 3.26 Å over 53 Cα, respectively). TyrRSMj possesses two additional regions not present in AlbC: a C-terminal domain involved in tRNA-binding and anticodon recognition coloured in grey, and a short N-terminal region coloured in light blue.
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Figure 3: Superimposition of the structures of AlbC and TyrRSMj in complex with its l-tyrosine substrate (PDB id: 1j1u). The two enzymes are shown in cartoon mode, and l-tyrosine is in ball and stick coloured in orange. The Rossmann-fold and the CP1 domains of TyrRSMj are coloured in dark and light blue, respectively, and the corresponding domains of AlbC are coloured in dark and light green, respectively. The Rossmann-fold and CP1 domains of the two proteins have similar rmsd values (3.25 Å over 96 Cα and 3.26 Å over 53 Cα, respectively). TyrRSMj possesses two additional regions not present in AlbC: a C-terminal domain involved in tRNA-binding and anticodon recognition coloured in grey, and a short N-terminal region coloured in light blue.

Mentions: We used the Dali server (28) to compare the structure of AlbC to protein structures in the PDB. Due to the Rossmann-fold domain, AlbC was found to share structural similarity with more than 300 proteins with a high Z-score (Z > 4) (Supplementary Table S1). Most of these proteins are class-I aaRSs, enzymes that catalyse the activation of amino acids and their transfer to cognate tRNAs to form aa-tRNAs. The 120 solutions with the highest Z-scores, from 10.7 to 7.3, were all obtained with the closely related class-Ic TyrRSs and TrpRSs, as previously reported for Rv2275 (12). The greatest similarity is with the archaeal Archaeglobus fulgidus (PDB id: 2cyb) and Methanococcus jannaschii TyrRSs (TyrRSMj) (PDB id: 1zh6, 1j1u), and with the eukaryotic Entamoeba histolytica TrpRS (TrpRSEh) (PDB id: 3hzr) (Supplementary Table S1). The 3D superimposition of AlbC and TyrRSMj (rmsd value of 3.5 Å over 160 Cα) (Figure 3) or TrpRSEh (rmsd value of 3.0 Å over 163 Cα) (Supplementary Figure S4) revealed that AlbC superimposes well with the N-terminal catalytic domains of the two aaRSs, despite low-sequence similarity (AlbC shares 15.5 and 15.8% similarity with the catalytic domains of TyrRSMj and TrpRSEh, respectively). Note that these enzymes possess C-terminal tRNA-binding domains whereas AlbC does not. The catalytic domains of each of the two aaRSs are composed of a Rossmann-fold domain and a connective-polypeptide 1 (CP1) domain that are both found in AlbC. The overall position of the two domains is conserved between AlbC and the two aaRSs and is mediated by substantial conservation of residues at the interface between the two domains. Most secondary-structure elements are well conserved, and the central β-sheets are well superimposed, but the helices that pack against these sheets present slight differences in length and positioning (Figure 3; Supplementary Figure S4). Moreover, the pocket in AlbC and the aminoacyl binding pockets in the two aaRSs are positioned similarly relative to the β-sheet forming part of the Rossmann-fold domain (29–33).Figure 3.


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)

Superimposition of the structures of AlbC and TyrRSMj in complex with its l-tyrosine substrate (PDB id: 1j1u). The two enzymes are shown in cartoon mode, and l-tyrosine is in ball and stick coloured in orange. The Rossmann-fold and the CP1 domains of TyrRSMj are coloured in dark and light blue, respectively, and the corresponding domains of AlbC are coloured in dark and light green, respectively. The Rossmann-fold and CP1 domains of the two proteins have similar rmsd values (3.25 Å over 96 Cα and 3.26 Å over 53 Cα, respectively). TyrRSMj possesses two additional regions not present in AlbC: a C-terminal domain involved in tRNA-binding and anticodon recognition coloured in grey, and a short N-terminal region coloured in light blue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105412&req=5

Figure 3: Superimposition of the structures of AlbC and TyrRSMj in complex with its l-tyrosine substrate (PDB id: 1j1u). The two enzymes are shown in cartoon mode, and l-tyrosine is in ball and stick coloured in orange. The Rossmann-fold and the CP1 domains of TyrRSMj are coloured in dark and light blue, respectively, and the corresponding domains of AlbC are coloured in dark and light green, respectively. The Rossmann-fold and CP1 domains of the two proteins have similar rmsd values (3.25 Å over 96 Cα and 3.26 Å over 53 Cα, respectively). TyrRSMj possesses two additional regions not present in AlbC: a C-terminal domain involved in tRNA-binding and anticodon recognition coloured in grey, and a short N-terminal region coloured in light blue.
Mentions: We used the Dali server (28) to compare the structure of AlbC to protein structures in the PDB. Due to the Rossmann-fold domain, AlbC was found to share structural similarity with more than 300 proteins with a high Z-score (Z > 4) (Supplementary Table S1). Most of these proteins are class-I aaRSs, enzymes that catalyse the activation of amino acids and their transfer to cognate tRNAs to form aa-tRNAs. The 120 solutions with the highest Z-scores, from 10.7 to 7.3, were all obtained with the closely related class-Ic TyrRSs and TrpRSs, as previously reported for Rv2275 (12). The greatest similarity is with the archaeal Archaeglobus fulgidus (PDB id: 2cyb) and Methanococcus jannaschii TyrRSs (TyrRSMj) (PDB id: 1zh6, 1j1u), and with the eukaryotic Entamoeba histolytica TrpRS (TrpRSEh) (PDB id: 3hzr) (Supplementary Table S1). The 3D superimposition of AlbC and TyrRSMj (rmsd value of 3.5 Å over 160 Cα) (Figure 3) or TrpRSEh (rmsd value of 3.0 Å over 163 Cα) (Supplementary Figure S4) revealed that AlbC superimposes well with the N-terminal catalytic domains of the two aaRSs, despite low-sequence similarity (AlbC shares 15.5 and 15.8% similarity with the catalytic domains of TyrRSMj and TrpRSEh, respectively). Note that these enzymes possess C-terminal tRNA-binding domains whereas AlbC does not. The catalytic domains of each of the two aaRSs are composed of a Rossmann-fold domain and a connective-polypeptide 1 (CP1) domain that are both found in AlbC. The overall position of the two domains is conserved between AlbC and the two aaRSs and is mediated by substantial conservation of residues at the interface between the two domains. Most secondary-structure elements are well conserved, and the central β-sheets are well superimposed, but the helices that pack against these sheets present slight differences in length and positioning (Figure 3; Supplementary Figure S4). Moreover, the pocket in AlbC and the aminoacyl binding pockets in the two aaRSs are positioned similarly relative to the β-sheet forming part of the Rossmann-fold domain (29–33).Figure 3.

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