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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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Comparison of the AlbC and TyrRSMj pockets. (A) The tyrosine-binding pocket of TyrRSMj in complex with its l-tyrosine substrate. TyrRS residues and l-tyrosine are represented in ball and stick, and coloured in blue and orange, respectively, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (B) Superimposition of the TyrRS residues (A) with the corresponding AlbC residues. AlbC residues are represented in ball and stick, and coloured in green, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. Residues lining the two pockets superimpose with an rmsd value of 1.49 Å over 32 main chain atoms. (C) The AlbC pocket in the presence of the phenylalanyl moiety of a Phe-tRNAPhe substrate. The phenylalanyl moiety position is mimicked on that of the l-tyrosine in the pocket of TyrRSMj, as shown in (A) and (B), except that the hydroxyl group has been removed. The conserved residue S37 and the residue L200, which have no corresponding residues in TyrRS, are shown in pink, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (D and E) Site-directed mutagenesis study of AlbC. Cyclodipeptide-synthesizing activities are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. (D) cFL-synthesizing activities of the wild-type AlbC (in blue) and of each of the variants in which a residue conserved among CDPSs is substituted (in grey). (E) cFL- and cYL-synthesizing activities (in blue and yellow, respectively) of the wild-type AlbC and the variant L200N. The wild-type AlbC synthesizes 33.5 ± 2.5 mg l−1 of cFL and 4.9 ± 0.5 mg l−1 of cYL (Supplementary Figure S6).
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Figure 5: Comparison of the AlbC and TyrRSMj pockets. (A) The tyrosine-binding pocket of TyrRSMj in complex with its l-tyrosine substrate. TyrRS residues and l-tyrosine are represented in ball and stick, and coloured in blue and orange, respectively, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (B) Superimposition of the TyrRS residues (A) with the corresponding AlbC residues. AlbC residues are represented in ball and stick, and coloured in green, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. Residues lining the two pockets superimpose with an rmsd value of 1.49 Å over 32 main chain atoms. (C) The AlbC pocket in the presence of the phenylalanyl moiety of a Phe-tRNAPhe substrate. The phenylalanyl moiety position is mimicked on that of the l-tyrosine in the pocket of TyrRSMj, as shown in (A) and (B), except that the hydroxyl group has been removed. The conserved residue S37 and the residue L200, which have no corresponding residues in TyrRS, are shown in pink, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (D and E) Site-directed mutagenesis study of AlbC. Cyclodipeptide-synthesizing activities are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. (D) cFL-synthesizing activities of the wild-type AlbC (in blue) and of each of the variants in which a residue conserved among CDPSs is substituted (in grey). (E) cFL- and cYL-synthesizing activities (in blue and yellow, respectively) of the wild-type AlbC and the variant L200N. The wild-type AlbC synthesizes 33.5 ± 2.5 mg l−1 of cFL and 4.9 ± 0.5 mg l−1 of cYL (Supplementary Figure S6).

Mentions: Using 3D superimposition, we compared the pocket in AlbC to the conserved amino acid-binding pockets in TyrRSMj and TrpRSEh (29–33). For example, the AlbC pocket was compared to that of the TyrRSMj complexed with its tyrosine substrate (Figure 5), which forms a hydrogen bond network with the conserved residues Y32, Y151, Q155, D158 and Q173, and interacts with the conserved residue G34 (29–31) (PDB id: 1j1u) (Figure 5A). The two pockets are positioned similarly relative to the Rossmann-fold. Each pocket contains some residues that are highly conserved in the corresponding family and that are remarkably well superimposed: the AlbC residues G35, Y178 and E182 superimpose with the TyrRSMj residues G34, Y151 and Q155, the rmsd values on all atoms being 1.30, 1.29 and 1.95 Å, respectively (Figures 4 and 5B). These structural similarities suggest that the pocket of AlbC could accommodate the aminoacyl moiety of an aa-tRNA substrate in the same way that TyrRSs bind their tyrosine substrates. To test this possibility, we independently substituted each residue in AlbC suspected to interact with the aminoacyl moiety of the substrate by alanine and, where relevant, other amino acids. We produced the resulting variants in E. coli and compared their in vivo synthesis of cFL, the major cyclodipeptide produced by AlbC, to that of the wild-type enzyme (1). The substitution of Y178 with alanine resulted in an almost inactive variant (Y178A), and its substitution with phenylalanine gave a poorly active variant (Y178F), despite being produced in amounts similar to that of the wild-type enzyme (Figure 5D). The variants E182A and E182Q were inactive, and the variant E182D had a weak but detectable activity although its production was strongly affected. The variant G35A was poorly active despite being abundantly produced (Figure 5D). Thus, the hydroxyl group of Y178 and the carboxylate group of E182 are essential for AlbC activity, suggesting that these groups may mediate hydrogen bonds with the main chain amino group of the substrate, in a way similar to what is described for TyrRSMj. The conserved residue G35 is also essential for the accommodation of the CDPS substrate. The conserved residues Y32 and D158 in TyrRSMj form hydrogen bonds with the hydroxyl group of the tyrosine substrate (Figure 5A), and superimpose with the hydrophobic residues L33 and L185, respectively, in AlbC (Figure 5B). This is consistent with hydrophobic phenylalanyl or leucyl moieties in the AlbC substrates. To try to accommodate a tyrosyl-tRNATyr substrate in the AlbC pocket, we attempted to construct an L33Y/L185D AlbC variant, but the genetic construct was not expressed (Figure 5D).Figure 5.


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)

Comparison of the AlbC and TyrRSMj pockets. (A) The tyrosine-binding pocket of TyrRSMj in complex with its l-tyrosine substrate. TyrRS residues and l-tyrosine are represented in ball and stick, and coloured in blue and orange, respectively, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (B) Superimposition of the TyrRS residues (A) with the corresponding AlbC residues. AlbC residues are represented in ball and stick, and coloured in green, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. Residues lining the two pockets superimpose with an rmsd value of 1.49 Å over 32 main chain atoms. (C) The AlbC pocket in the presence of the phenylalanyl moiety of a Phe-tRNAPhe substrate. The phenylalanyl moiety position is mimicked on that of the l-tyrosine in the pocket of TyrRSMj, as shown in (A) and (B), except that the hydroxyl group has been removed. The conserved residue S37 and the residue L200, which have no corresponding residues in TyrRS, are shown in pink, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (D and E) Site-directed mutagenesis study of AlbC. Cyclodipeptide-synthesizing activities are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. (D) cFL-synthesizing activities of the wild-type AlbC (in blue) and of each of the variants in which a residue conserved among CDPSs is substituted (in grey). (E) cFL- and cYL-synthesizing activities (in blue and yellow, respectively) of the wild-type AlbC and the variant L200N. The wild-type AlbC synthesizes 33.5 ± 2.5 mg l−1 of cFL and 4.9 ± 0.5 mg l−1 of cYL (Supplementary Figure S6).
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Figure 5: Comparison of the AlbC and TyrRSMj pockets. (A) The tyrosine-binding pocket of TyrRSMj in complex with its l-tyrosine substrate. TyrRS residues and l-tyrosine are represented in ball and stick, and coloured in blue and orange, respectively, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (B) Superimposition of the TyrRS residues (A) with the corresponding AlbC residues. AlbC residues are represented in ball and stick, and coloured in green, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. Residues lining the two pockets superimpose with an rmsd value of 1.49 Å over 32 main chain atoms. (C) The AlbC pocket in the presence of the phenylalanyl moiety of a Phe-tRNAPhe substrate. The phenylalanyl moiety position is mimicked on that of the l-tyrosine in the pocket of TyrRSMj, as shown in (A) and (B), except that the hydroxyl group has been removed. The conserved residue S37 and the residue L200, which have no corresponding residues in TyrRS, are shown in pink, with oxygen and nitrogen atoms coloured in red and dark blue, respectively. (D and E) Site-directed mutagenesis study of AlbC. Cyclodipeptide-synthesizing activities are shown with error bars. The corresponding western blots indicating amounts of the proteins are also shown. (D) cFL-synthesizing activities of the wild-type AlbC (in blue) and of each of the variants in which a residue conserved among CDPSs is substituted (in grey). (E) cFL- and cYL-synthesizing activities (in blue and yellow, respectively) of the wild-type AlbC and the variant L200N. The wild-type AlbC synthesizes 33.5 ± 2.5 mg l−1 of cFL and 4.9 ± 0.5 mg l−1 of cYL (Supplementary Figure S6).
Mentions: Using 3D superimposition, we compared the pocket in AlbC to the conserved amino acid-binding pockets in TyrRSMj and TrpRSEh (29–33). For example, the AlbC pocket was compared to that of the TyrRSMj complexed with its tyrosine substrate (Figure 5), which forms a hydrogen bond network with the conserved residues Y32, Y151, Q155, D158 and Q173, and interacts with the conserved residue G34 (29–31) (PDB id: 1j1u) (Figure 5A). The two pockets are positioned similarly relative to the Rossmann-fold. Each pocket contains some residues that are highly conserved in the corresponding family and that are remarkably well superimposed: the AlbC residues G35, Y178 and E182 superimpose with the TyrRSMj residues G34, Y151 and Q155, the rmsd values on all atoms being 1.30, 1.29 and 1.95 Å, respectively (Figures 4 and 5B). These structural similarities suggest that the pocket of AlbC could accommodate the aminoacyl moiety of an aa-tRNA substrate in the same way that TyrRSs bind their tyrosine substrates. To test this possibility, we independently substituted each residue in AlbC suspected to interact with the aminoacyl moiety of the substrate by alanine and, where relevant, other amino acids. We produced the resulting variants in E. coli and compared their in vivo synthesis of cFL, the major cyclodipeptide produced by AlbC, to that of the wild-type enzyme (1). The substitution of Y178 with alanine resulted in an almost inactive variant (Y178A), and its substitution with phenylalanine gave a poorly active variant (Y178F), despite being produced in amounts similar to that of the wild-type enzyme (Figure 5D). The variants E182A and E182Q were inactive, and the variant E182D had a weak but detectable activity although its production was strongly affected. The variant G35A was poorly active despite being abundantly produced (Figure 5D). Thus, the hydroxyl group of Y178 and the carboxylate group of E182 are essential for AlbC activity, suggesting that these groups may mediate hydrogen bonds with the main chain amino group of the substrate, in a way similar to what is described for TyrRSMj. The conserved residue G35 is also essential for the accommodation of the CDPS substrate. The conserved residues Y32 and D158 in TyrRSMj form hydrogen bonds with the hydroxyl group of the tyrosine substrate (Figure 5A), and superimpose with the hydrophobic residues L33 and L185, respectively, in AlbC (Figure 5B). This is consistent with hydrophobic phenylalanyl or leucyl moieties in the AlbC substrates. To try to accommodate a tyrosyl-tRNATyr substrate in the AlbC pocket, we attempted to construct an L33Y/L185D AlbC variant, but the genetic construct was not expressed (Figure 5D).Figure 5.

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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