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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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Basic patch residues contributing to CDPS activity. (A) Electrostatic surface potential of AlbC, mapped on its solvent-accessible surface at contouring levels of ±5 kTe–1. Positive charge is in blue, negative charge in red. The potential was calculated using APBS in PyMol (59) (http://www.pymol.org/) from a derived model of AlbC where the missing side chain of residue R215 and the missing residue T217 were added. AlbC structure is shown in grey and the side chains of all mutated basic residues are identified as sticks. (B) Electrostatic surface potential of Rv2275 generated like that of AlbC. (C) Site-directed mutagenesis study of AlbC. cFL-synthesizing activity of the wild-type AlbC (in blue) and of each of the variants in which a basic residue is substituted (in grey) are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. Left, the residues belonging to the helix α4; right, other basic residues.
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Figure 8: Basic patch residues contributing to CDPS activity. (A) Electrostatic surface potential of AlbC, mapped on its solvent-accessible surface at contouring levels of ±5 kTe–1. Positive charge is in blue, negative charge in red. The potential was calculated using APBS in PyMol (59) (http://www.pymol.org/) from a derived model of AlbC where the missing side chain of residue R215 and the missing residue T217 were added. AlbC structure is shown in grey and the side chains of all mutated basic residues are identified as sticks. (B) Electrostatic surface potential of Rv2275 generated like that of AlbC. (C) Site-directed mutagenesis study of AlbC. cFL-synthesizing activity of the wild-type AlbC (in blue) and of each of the variants in which a basic residue is substituted (in grey) are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. Left, the residues belonging to the helix α4; right, other basic residues.

Mentions: The electrostatic potential surface of AlbC presents a highly biased distribution of charged residues, which could interact with the tRNA substrate by forming salt bridges with the phosphates of the tRNA backbone or hydrogen bonds with the nucleotide bases. In particular, there is a large patch of positively charged residues covering a surface of 1040 Å2 (Figure 8A). This patch is mostly composed of residues belonging to the helix α4, which contains nine basic residues. These residues, R87, K90, R91, K94, R97, R98, R99, R101 and R102, protrude toward the solvent, and a similar pattern of basic residues is found in all CDPSs (Figures 4 and 8B). We substituted with alanine each of the basic residues in helix α4. All the resulting variants were produced with yields similar to that of the wild-type enzyme but their cFL-synthesizing activities were diverse (Figure 8C). The variants R98A, R99A and R98A/R99A displayed only 19, 8 and <2% of the wild-type activity, respectively. Five other variants (K90A, K94A, R97A, R101A and R102A) had activities that were 53–72% of the wild-type enzyme. The activities of the two remaining variants, R87A and R91A, were similar to that of the wild type. These results showed that the basic residues located in the C-terminal half of the helix α4, especially R98 and R99, are important for cFL production, probably because they interact with the tRNA moiety of the substrate. A second patch of basic residues, consisting of R214, R215 and R220 from loop α8–β7 and R231 from loop β7–α9, is present on AlbC (Figure 8A). We substituted these residues and showed that the corresponding variants displayed properties similar to the wild-type enzyme (Figure 8C), indicating that this patch is not involved in tRNA binding. This is consistent with the fact that this patch is not conserved among CDPSs (Figure 4). Our results suggest that the interaction between AlbC and its aa-tRNA is driven by the binding of the substrate aminoacyl moiety in its deep pocket and the interaction of the tRNA moiety with at least one patch of basic residues on helix α4.Figure 8.


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)

Basic patch residues contributing to CDPS activity. (A) Electrostatic surface potential of AlbC, mapped on its solvent-accessible surface at contouring levels of ±5 kTe–1. Positive charge is in blue, negative charge in red. The potential was calculated using APBS in PyMol (59) (http://www.pymol.org/) from a derived model of AlbC where the missing side chain of residue R215 and the missing residue T217 were added. AlbC structure is shown in grey and the side chains of all mutated basic residues are identified as sticks. (B) Electrostatic surface potential of Rv2275 generated like that of AlbC. (C) Site-directed mutagenesis study of AlbC. cFL-synthesizing activity of the wild-type AlbC (in blue) and of each of the variants in which a basic residue is substituted (in grey) are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. Left, the residues belonging to the helix α4; right, other basic residues.
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Related In: Results  -  Collection

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Figure 8: Basic patch residues contributing to CDPS activity. (A) Electrostatic surface potential of AlbC, mapped on its solvent-accessible surface at contouring levels of ±5 kTe–1. Positive charge is in blue, negative charge in red. The potential was calculated using APBS in PyMol (59) (http://www.pymol.org/) from a derived model of AlbC where the missing side chain of residue R215 and the missing residue T217 were added. AlbC structure is shown in grey and the side chains of all mutated basic residues are identified as sticks. (B) Electrostatic surface potential of Rv2275 generated like that of AlbC. (C) Site-directed mutagenesis study of AlbC. cFL-synthesizing activity of the wild-type AlbC (in blue) and of each of the variants in which a basic residue is substituted (in grey) are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. Left, the residues belonging to the helix α4; right, other basic residues.
Mentions: The electrostatic potential surface of AlbC presents a highly biased distribution of charged residues, which could interact with the tRNA substrate by forming salt bridges with the phosphates of the tRNA backbone or hydrogen bonds with the nucleotide bases. In particular, there is a large patch of positively charged residues covering a surface of 1040 Å2 (Figure 8A). This patch is mostly composed of residues belonging to the helix α4, which contains nine basic residues. These residues, R87, K90, R91, K94, R97, R98, R99, R101 and R102, protrude toward the solvent, and a similar pattern of basic residues is found in all CDPSs (Figures 4 and 8B). We substituted with alanine each of the basic residues in helix α4. All the resulting variants were produced with yields similar to that of the wild-type enzyme but their cFL-synthesizing activities were diverse (Figure 8C). The variants R98A, R99A and R98A/R99A displayed only 19, 8 and <2% of the wild-type activity, respectively. Five other variants (K90A, K94A, R97A, R101A and R102A) had activities that were 53–72% of the wild-type enzyme. The activities of the two remaining variants, R87A and R91A, were similar to that of the wild type. These results showed that the basic residues located in the C-terminal half of the helix α4, especially R98 and R99, are important for cFL production, probably because they interact with the tRNA moiety of the substrate. A second patch of basic residues, consisting of R214, R215 and R220 from loop α8–β7 and R231 from loop β7–α9, is present on AlbC (Figure 8A). We substituted these residues and showed that the corresponding variants displayed properties similar to the wild-type enzyme (Figure 8C), indicating that this patch is not involved in tRNA binding. This is consistent with the fact that this patch is not conserved among CDPSs (Figure 4). Our results suggest that the interaction between AlbC and its aa-tRNA is driven by the binding of the substrate aminoacyl moiety in its deep pocket and the interaction of the tRNA moiety with at least one patch of basic residues on helix α4.Figure 8.

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