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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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Structure-based sequence alignment of six CDPSs and three TyrRSs from the three primary kingdoms. The secondary-structural elements of AlbC and the TyrRSMj are indicated above and below the alignment, respectively. The Rossmann-fold and the CP1 domains of AlbC are shown in dark and light green, respectively. The N-terminal region, the Rossmann-fold domain, and the CP1 domain of the TyrRSMj are shown in yellow, dark and light blue, respectively. The core regions of AlbC and the TyrRSMj displaying greatest structural similarity (rmsd value of 2.36 Å over 116 Cα) are shown with a gray background. The residues conserved among CDPSs, and those conserved among TyrRSs are indicated by a red background. Black stars above the AlbC sequence indicate the residues that were modified by site-directed mutagenesis in this study. The basic residues of CDPSs, which define a patch of positively charged residues belonging to the helix α4, are indicated by an orange background. The residues that are disordered in the crystal structure of AlbC and Rv2275 are shown in grey. The residues of TyrRSs previously shown to be involved in tyrosine recognition are indicated in dark blue; the position of the two clusters involved in acceptor stem recognition (41) are indicated by pink lines below the TyrRSs sequences, and the residues involved in this recognition are indicated by a pink background. Black lines below the TyrRSs sequences indicate the class I signature motifs, HIGH and KMSKS, which are involved in ATP binding. Rv2275 residues that are involved in the interface of the dimer are shown with a green background. Protein sequence alignments were done according to structural alignments for AlbC and TyrRSs and were corrected manually for CDPS based on previously described alignments (1). Two CDPSs are not mentioned, namely YvmC from Bacillus thuringiensis and YvmC from B. licheniformis, because they are very close to YvmC from B. subtilis. Abbreviations: Bsub, B. subtilis; BacSt, Geobacillus stearothermophilus; MetJa, M. jannaschii.
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Figure 4: Structure-based sequence alignment of six CDPSs and three TyrRSs from the three primary kingdoms. The secondary-structural elements of AlbC and the TyrRSMj are indicated above and below the alignment, respectively. The Rossmann-fold and the CP1 domains of AlbC are shown in dark and light green, respectively. The N-terminal region, the Rossmann-fold domain, and the CP1 domain of the TyrRSMj are shown in yellow, dark and light blue, respectively. The core regions of AlbC and the TyrRSMj displaying greatest structural similarity (rmsd value of 2.36 Å over 116 Cα) are shown with a gray background. The residues conserved among CDPSs, and those conserved among TyrRSs are indicated by a red background. Black stars above the AlbC sequence indicate the residues that were modified by site-directed mutagenesis in this study. The basic residues of CDPSs, which define a patch of positively charged residues belonging to the helix α4, are indicated by an orange background. The residues that are disordered in the crystal structure of AlbC and Rv2275 are shown in grey. The residues of TyrRSs previously shown to be involved in tyrosine recognition are indicated in dark blue; the position of the two clusters involved in acceptor stem recognition (41) are indicated by pink lines below the TyrRSs sequences, and the residues involved in this recognition are indicated by a pink background. Black lines below the TyrRSs sequences indicate the class I signature motifs, HIGH and KMSKS, which are involved in ATP binding. Rv2275 residues that are involved in the interface of the dimer are shown with a green background. Protein sequence alignments were done according to structural alignments for AlbC and TyrRSs and were corrected manually for CDPS based on previously described alignments (1). Two CDPSs are not mentioned, namely YvmC from Bacillus thuringiensis and YvmC from B. licheniformis, because they are very close to YvmC from B. subtilis. Abbreviations: Bsub, B. subtilis; BacSt, Geobacillus stearothermophilus; MetJa, M. jannaschii.

Mentions: However, there are some major differences between AlbC and the two aaRSs. The hydrophobic regions of the CP1 domains of TyrRS or TrpRS involved in the homodimerization of these enzymes (34,35) are not found in AlbC. In addition, when we mimicked this association for two AlbC monomers by superimposing them on the crystal structure of TyrRSMj, we did not find any complementary dimer interface, and we observed major steric hindrances between helices α5, α6 and α7 of each AlbC monomer (Supplementary Figure S5A). However, the crystal structure of Rv2275 was obtained as a homodimer. But, the Rv2275 residues that make up the interface belong to other secondary-structural elements than those involved in the homodimerization of the two aaRSs (12). Noted that these residues are not conserved in CDPSs (Figure 4 and Supplementary Figure S5B). All these observations are consistent with AlbC being found as a monomer both in solution (1) and in crystal forms. Another major difference between CDPSs and class-I aaRSs is that CDPSs do not have the two consensus motifs that are conserved in class-I aaRSs and are involved in ATP binding (36–38) (Figure 4).Figure 4.


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)

Structure-based sequence alignment of six CDPSs and three TyrRSs from the three primary kingdoms. The secondary-structural elements of AlbC and the TyrRSMj are indicated above and below the alignment, respectively. The Rossmann-fold and the CP1 domains of AlbC are shown in dark and light green, respectively. The N-terminal region, the Rossmann-fold domain, and the CP1 domain of the TyrRSMj are shown in yellow, dark and light blue, respectively. The core regions of AlbC and the TyrRSMj displaying greatest structural similarity (rmsd value of 2.36 Å over 116 Cα) are shown with a gray background. The residues conserved among CDPSs, and those conserved among TyrRSs are indicated by a red background. Black stars above the AlbC sequence indicate the residues that were modified by site-directed mutagenesis in this study. The basic residues of CDPSs, which define a patch of positively charged residues belonging to the helix α4, are indicated by an orange background. The residues that are disordered in the crystal structure of AlbC and Rv2275 are shown in grey. The residues of TyrRSs previously shown to be involved in tyrosine recognition are indicated in dark blue; the position of the two clusters involved in acceptor stem recognition (41) are indicated by pink lines below the TyrRSs sequences, and the residues involved in this recognition are indicated by a pink background. Black lines below the TyrRSs sequences indicate the class I signature motifs, HIGH and KMSKS, which are involved in ATP binding. Rv2275 residues that are involved in the interface of the dimer are shown with a green background. Protein sequence alignments were done according to structural alignments for AlbC and TyrRSs and were corrected manually for CDPS based on previously described alignments (1). Two CDPSs are not mentioned, namely YvmC from Bacillus thuringiensis and YvmC from B. licheniformis, because they are very close to YvmC from B. subtilis. Abbreviations: Bsub, B. subtilis; BacSt, Geobacillus stearothermophilus; MetJa, M. jannaschii.
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Figure 4: Structure-based sequence alignment of six CDPSs and three TyrRSs from the three primary kingdoms. The secondary-structural elements of AlbC and the TyrRSMj are indicated above and below the alignment, respectively. The Rossmann-fold and the CP1 domains of AlbC are shown in dark and light green, respectively. The N-terminal region, the Rossmann-fold domain, and the CP1 domain of the TyrRSMj are shown in yellow, dark and light blue, respectively. The core regions of AlbC and the TyrRSMj displaying greatest structural similarity (rmsd value of 2.36 Å over 116 Cα) are shown with a gray background. The residues conserved among CDPSs, and those conserved among TyrRSs are indicated by a red background. Black stars above the AlbC sequence indicate the residues that were modified by site-directed mutagenesis in this study. The basic residues of CDPSs, which define a patch of positively charged residues belonging to the helix α4, are indicated by an orange background. The residues that are disordered in the crystal structure of AlbC and Rv2275 are shown in grey. The residues of TyrRSs previously shown to be involved in tyrosine recognition are indicated in dark blue; the position of the two clusters involved in acceptor stem recognition (41) are indicated by pink lines below the TyrRSs sequences, and the residues involved in this recognition are indicated by a pink background. Black lines below the TyrRSs sequences indicate the class I signature motifs, HIGH and KMSKS, which are involved in ATP binding. Rv2275 residues that are involved in the interface of the dimer are shown with a green background. Protein sequence alignments were done according to structural alignments for AlbC and TyrRSs and were corrected manually for CDPS based on previously described alignments (1). Two CDPSs are not mentioned, namely YvmC from Bacillus thuringiensis and YvmC from B. licheniformis, because they are very close to YvmC from B. subtilis. Abbreviations: Bsub, B. subtilis; BacSt, Geobacillus stearothermophilus; MetJa, M. jannaschii.
Mentions: However, there are some major differences between AlbC and the two aaRSs. The hydrophobic regions of the CP1 domains of TyrRS or TrpRS involved in the homodimerization of these enzymes (34,35) are not found in AlbC. In addition, when we mimicked this association for two AlbC monomers by superimposing them on the crystal structure of TyrRSMj, we did not find any complementary dimer interface, and we observed major steric hindrances between helices α5, α6 and α7 of each AlbC monomer (Supplementary Figure S5A). However, the crystal structure of Rv2275 was obtained as a homodimer. But, the Rv2275 residues that make up the interface belong to other secondary-structural elements than those involved in the homodimerization of the two aaRSs (12). Noted that these residues are not conserved in CDPSs (Figure 4 and Supplementary Figure S5B). All these observations are consistent with AlbC being found as a monomer both in solution (1) and in crystal forms. Another major difference between CDPSs and class-I aaRSs is that CDPSs do not have the two consensus motifs that are conserved in class-I aaRSs and are involved in ATP binding (36–38) (Figure 4).Figure 4.

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