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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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Proposed mechanism of covalent phenylalanyl-enzyme formation for AlbC. (A) Proposed anchoring of the Phe-tRNAPhe substrate at the AlbC active site. The residues S37, Y178 and E182, which are essential for the covalent intermediate formation (Figures 6 and 7), are represented in green. The residue L200, shown to interact with the phenylalanyl moiety of the substrate (Figure 5E), is also represented in green. As no residue in the AlbC active site is likely to deprotonate the hydroxyl group of S37, the S37 activation is proposed to be achieved by a concerted proton shuttling mechanism involving the two adjacent vicinal hydroxyls of the nucleotide A76 of the tRNA moiety. (B) The resulting covalent phenylalanyl-enzyme.
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Figure 9: Proposed mechanism of covalent phenylalanyl-enzyme formation for AlbC. (A) Proposed anchoring of the Phe-tRNAPhe substrate at the AlbC active site. The residues S37, Y178 and E182, which are essential for the covalent intermediate formation (Figures 6 and 7), are represented in green. The residue L200, shown to interact with the phenylalanyl moiety of the substrate (Figure 5E), is also represented in green. As no residue in the AlbC active site is likely to deprotonate the hydroxyl group of S37, the S37 activation is proposed to be achieved by a concerted proton shuttling mechanism involving the two adjacent vicinal hydroxyls of the nucleotide A76 of the tRNA moiety. (B) The resulting covalent phenylalanyl-enzyme.

Mentions: We wondered how AlbC uses two tRNA substrates to form cyclodipeptides. One possibility is that AlbC acts as a homodimer, each monomer interacting with one aa-tRNA. Although AlbC was obtained as a monomer both in solution (1) and in crystal forms, we cannot exclude that aa-tRNA binding induces dimerization. Noted that class-Ic aaRSs act as functional dimers, but they are also obtained as dimers in crystals (44). Class-Ic aaRSs dimers are mainly formed by the association of a hydrophobic surface involving residues of the CP1 domain of each catalytic domain (34,35). The positions of these residues are not conserved in AlbC. In addition, when we mimicked this association for two AlbC monomers by superimposing them on different crystal structures of TyrRSs or TrpRSs, we observed major steric hindrances between helices α5, α6 and α7 of each AlbC monomer. Therefore, it is unlikely that AlbC dimerizes in a manner similar to class-Ic aaRSs, and furthermore, any such dimerization would result in a physical separation of the two AlbC catalytic pockets, inconsistent with the formation of two peptide bonds. This is indeed what was observed for Rv2275, which is found as a homodimer: the residues that make up the interface belong to other secondary structural elements than those involved in the dimerization of class-Ic aaRSs, and the two Rv2275 catalytic pockets are not at the dimer interface, but are physically separated (12). Moreover, it is unlikely that AlbC dimerizes in a manner similar to Rv2275 since the residues that are involved in the Rv2275 interface are not conserved in AlbC and other CDPSs. As it seems more probable that AlbC is active as a monomer, we considered whether AlbC catalyses its two-substrate reaction using a ternary complex or a ping-pong mechanism. The size of the AlbC pocket does not appear appropriate to accommodate the aminoacyl moieties of two aa-tRNA substrates concomitantly. Thus, one aa-tRNA may bind to AlbC and its aminoacyl moiety would be stored as a covalent intermediate, i.e. as an aminoacyl-enzyme intermediate. The formation of a covalent intermediate resulting from the transfer of l-Tyr from Tyr-tRNATyr to an active serine was suggested for Rv2275, but such a transfer was not conclusively demonstrated (12). We demonstrated that this transfer occurs in AlbC because we showed that the active serine residue is covalently bound to the phenylalanyl transferred from Phe-tRNAPhe during the catalytic cycle. An intriguing point that remains to be determined concerns the activation of the catalytic serine, i.e. the deprotonation of its hydroxyl group serving as the nucleophile. For Rv2275, it was suggested that the catalytic serine (S88) residue is activated by a conserved tyrosine residue (Y253). This is consistent with the hydrogen bonding of Y253 to the side chain hydroxyl group of S88, and the much lower (by a factor of 200) catalytic activity of the Y253F variant (12). For AlbC, the corresponding tyrosine residue (Y202) adopts a different conformation in the AlbC structure, and the Y202F variant displays only a 10-fold decrease in catalytic activity. We also unambiguously showed that the formation of the covalent aminoacyl intermediate is not affected by the substitution of Y202 with phenylalanine, demonstrating that Y202 is not responsible for the activation of S37 in AlbC. As no other residue in the vicinity of S37 can account for its activation, an attractive hypothesis is that the serine activation results from a concerted proton shuttling mechanism involving the two adjacent vicinal hydroxyls of the nucleotide A76 of the tRNA moiety (Figure 9), as it has been proposed for the ribosomal peptididyl transferase (48) and the FemX aa-transferase of Weissella viridescens (49). At this stage, we cannot exclude some rearrangements in AlbC active site during the different steps of the reaction. The analysis of B-factors around AlbC active site suggests that the loop β3–α2, which precedes the position S37, may present some flexibility. The crystal structure of AlbC complexed with different intermediates of the reaction should clarify this point. But, whatever how the serine activation is performed, we demonstrated that CDPSs use a ping-pong mechanism involving a covalent aminoacyl-enzyme intermediate. The aminoacyl-enzyme may then react with the aminoacyl moiety of the second aa-tRNA to form a dipeptidyl-enzyme or dipeptidyl-tRNA intermediate, which may then undergo intramolecular cyclization leading to the formation of the cyclodipeptide product.Figure 9.


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)

Proposed mechanism of covalent phenylalanyl-enzyme formation for AlbC. (A) Proposed anchoring of the Phe-tRNAPhe substrate at the AlbC active site. The residues S37, Y178 and E182, which are essential for the covalent intermediate formation (Figures 6 and 7), are represented in green. The residue L200, shown to interact with the phenylalanyl moiety of the substrate (Figure 5E), is also represented in green. As no residue in the AlbC active site is likely to deprotonate the hydroxyl group of S37, the S37 activation is proposed to be achieved by a concerted proton shuttling mechanism involving the two adjacent vicinal hydroxyls of the nucleotide A76 of the tRNA moiety. (B) The resulting covalent phenylalanyl-enzyme.
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Figure 9: Proposed mechanism of covalent phenylalanyl-enzyme formation for AlbC. (A) Proposed anchoring of the Phe-tRNAPhe substrate at the AlbC active site. The residues S37, Y178 and E182, which are essential for the covalent intermediate formation (Figures 6 and 7), are represented in green. The residue L200, shown to interact with the phenylalanyl moiety of the substrate (Figure 5E), is also represented in green. As no residue in the AlbC active site is likely to deprotonate the hydroxyl group of S37, the S37 activation is proposed to be achieved by a concerted proton shuttling mechanism involving the two adjacent vicinal hydroxyls of the nucleotide A76 of the tRNA moiety. (B) The resulting covalent phenylalanyl-enzyme.
Mentions: We wondered how AlbC uses two tRNA substrates to form cyclodipeptides. One possibility is that AlbC acts as a homodimer, each monomer interacting with one aa-tRNA. Although AlbC was obtained as a monomer both in solution (1) and in crystal forms, we cannot exclude that aa-tRNA binding induces dimerization. Noted that class-Ic aaRSs act as functional dimers, but they are also obtained as dimers in crystals (44). Class-Ic aaRSs dimers are mainly formed by the association of a hydrophobic surface involving residues of the CP1 domain of each catalytic domain (34,35). The positions of these residues are not conserved in AlbC. In addition, when we mimicked this association for two AlbC monomers by superimposing them on different crystal structures of TyrRSs or TrpRSs, we observed major steric hindrances between helices α5, α6 and α7 of each AlbC monomer. Therefore, it is unlikely that AlbC dimerizes in a manner similar to class-Ic aaRSs, and furthermore, any such dimerization would result in a physical separation of the two AlbC catalytic pockets, inconsistent with the formation of two peptide bonds. This is indeed what was observed for Rv2275, which is found as a homodimer: the residues that make up the interface belong to other secondary structural elements than those involved in the dimerization of class-Ic aaRSs, and the two Rv2275 catalytic pockets are not at the dimer interface, but are physically separated (12). Moreover, it is unlikely that AlbC dimerizes in a manner similar to Rv2275 since the residues that are involved in the Rv2275 interface are not conserved in AlbC and other CDPSs. As it seems more probable that AlbC is active as a monomer, we considered whether AlbC catalyses its two-substrate reaction using a ternary complex or a ping-pong mechanism. The size of the AlbC pocket does not appear appropriate to accommodate the aminoacyl moieties of two aa-tRNA substrates concomitantly. Thus, one aa-tRNA may bind to AlbC and its aminoacyl moiety would be stored as a covalent intermediate, i.e. as an aminoacyl-enzyme intermediate. The formation of a covalent intermediate resulting from the transfer of l-Tyr from Tyr-tRNATyr to an active serine was suggested for Rv2275, but such a transfer was not conclusively demonstrated (12). We demonstrated that this transfer occurs in AlbC because we showed that the active serine residue is covalently bound to the phenylalanyl transferred from Phe-tRNAPhe during the catalytic cycle. An intriguing point that remains to be determined concerns the activation of the catalytic serine, i.e. the deprotonation of its hydroxyl group serving as the nucleophile. For Rv2275, it was suggested that the catalytic serine (S88) residue is activated by a conserved tyrosine residue (Y253). This is consistent with the hydrogen bonding of Y253 to the side chain hydroxyl group of S88, and the much lower (by a factor of 200) catalytic activity of the Y253F variant (12). For AlbC, the corresponding tyrosine residue (Y202) adopts a different conformation in the AlbC structure, and the Y202F variant displays only a 10-fold decrease in catalytic activity. We also unambiguously showed that the formation of the covalent aminoacyl intermediate is not affected by the substitution of Y202 with phenylalanine, demonstrating that Y202 is not responsible for the activation of S37 in AlbC. As no other residue in the vicinity of S37 can account for its activation, an attractive hypothesis is that the serine activation results from a concerted proton shuttling mechanism involving the two adjacent vicinal hydroxyls of the nucleotide A76 of the tRNA moiety (Figure 9), as it has been proposed for the ribosomal peptididyl transferase (48) and the FemX aa-transferase of Weissella viridescens (49). At this stage, we cannot exclude some rearrangements in AlbC active site during the different steps of the reaction. The analysis of B-factors around AlbC active site suggests that the loop β3–α2, which precedes the position S37, may present some flexibility. The crystal structure of AlbC complexed with different intermediates of the reaction should clarify this point. But, whatever how the serine activation is performed, we demonstrated that CDPSs use a ping-pong mechanism involving a covalent aminoacyl-enzyme intermediate. The aminoacyl-enzyme may then react with the aminoacyl moiety of the second aa-tRNA to form a dipeptidyl-enzyme or dipeptidyl-tRNA intermediate, which may then undergo intramolecular cyclization leading to the formation of the cyclodipeptide product.Figure 9.

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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