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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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Covalent labelling of AlbC and variants by tritiated Phe transferred from [3H]Phe-tRNAPhe. Enzymes were incubated with [3H]Phe-tRNAPhe, as described in ‘Materials and Methods’ section, separated on SDS–PAGE, then transferred onto a PVDF membrane that was analysed with a radioimager.
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Figure 6: Covalent labelling of AlbC and variants by tritiated Phe transferred from [3H]Phe-tRNAPhe. Enzymes were incubated with [3H]Phe-tRNAPhe, as described in ‘Materials and Methods’ section, separated on SDS–PAGE, then transferred onto a PVDF membrane that was analysed with a radioimager.

Mentions: The superimposition of the pockets of AlbC and TyrRSMj revealed significant similarities (see above), but also intriguing differences. The strictly conserved residue S37 of AlbC has no corresponding conserved residue in class Ic-aaRSs (Figures 4 and 5B). The position of the phenylalanyl moiety of a Phe-tRNAPhe in the AlbC pocket, as predicted by analogy with the tyrosine substrate in the TyrRSMj pocket (Figure 5C), indicates that the Oγ atom of S37 would be located in the proximity of the carbonyl carbon of the esterified Phe-tRNAPhe substrate. This organization together with the catalysed reaction—the formation of a peptide bond from aminoacyl-tRNA—suggests that S37 may be a catalytic residue serving as a nucleophile. This is consistent with the fact that substitution of S37 with alanine resulted in an inactive variant (S37A), but its substitution with the nucleophilic cysteine (S37C) did not completely abolish the enzymatic activity (Figure 5D). So, S37 may attack the carbonyl carbon of the esterified Phe-tRNAPhe substrate, leading to the covalent phenylalanylation of S37. The formation of a covalent intermediate in which l-Tyr is transferred from Tyr-tRNATyr to an active serine (S88) was also suggested for Rv2275, using radioactive labelling (12). Moreover, the S88 residue of Rv2275 was suggested to be activated by the conserved Y253 residue, which corresponds to Y202 in AlbC (12). To verify the existence of a covalent intermediate, we further analysed AlbC and the five variants S37A, S37C, Y178F, E182Q and Y202F. The enzymes were incubated in the presence of [3H]Phe-tRNAPhe, and they were separated by SDS–PAGE before being transferred on a PVDF membrane, which was analysed with a radioimager. As observed for Rv2275, wild-type AlbC retained a high amount of the radiolabel, whereas the variant S37A did not retain any radiolabel. The variant S37C presented behaviour similar to that of the wild-type enzyme, confirming the formation of a covalent intermediate and the probable status of S37 as the point of covalent attachment (Figure 6). The variants Y178F and E182Q retained significant amounts of the radiolabel although much less than the wild-type enzyme (Figure 6). This result, combined with the substantial and complete loss of enzyme activity for the variants Y178F and E182Q, respectively (Figure 5D), argues that Y178 and E182 may play a crucial role in the good positioning of the aminoacyl moiety of the substrate in the active site—essential for an efficient formation of the covalent intermediate—concerning the variant Y202F, it retained ∼11% of the enzymatic activity (Figure 5D), and at least as much radiolabel as the wild-type enzyme (Figure 6). As the removal of the hydroxyl group in the variant Y202F does not prevent the formation of the covalent intermediate, this demonstrated that Y202 is not responsible for the S37 activation in AlbC.Figure 6.


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)

Covalent labelling of AlbC and variants by tritiated Phe transferred from [3H]Phe-tRNAPhe. Enzymes were incubated with [3H]Phe-tRNAPhe, as described in ‘Materials and Methods’ section, separated on SDS–PAGE, then transferred onto a PVDF membrane that was analysed with a radioimager.
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Figure 6: Covalent labelling of AlbC and variants by tritiated Phe transferred from [3H]Phe-tRNAPhe. Enzymes were incubated with [3H]Phe-tRNAPhe, as described in ‘Materials and Methods’ section, separated on SDS–PAGE, then transferred onto a PVDF membrane that was analysed with a radioimager.
Mentions: The superimposition of the pockets of AlbC and TyrRSMj revealed significant similarities (see above), but also intriguing differences. The strictly conserved residue S37 of AlbC has no corresponding conserved residue in class Ic-aaRSs (Figures 4 and 5B). The position of the phenylalanyl moiety of a Phe-tRNAPhe in the AlbC pocket, as predicted by analogy with the tyrosine substrate in the TyrRSMj pocket (Figure 5C), indicates that the Oγ atom of S37 would be located in the proximity of the carbonyl carbon of the esterified Phe-tRNAPhe substrate. This organization together with the catalysed reaction—the formation of a peptide bond from aminoacyl-tRNA—suggests that S37 may be a catalytic residue serving as a nucleophile. This is consistent with the fact that substitution of S37 with alanine resulted in an inactive variant (S37A), but its substitution with the nucleophilic cysteine (S37C) did not completely abolish the enzymatic activity (Figure 5D). So, S37 may attack the carbonyl carbon of the esterified Phe-tRNAPhe substrate, leading to the covalent phenylalanylation of S37. The formation of a covalent intermediate in which l-Tyr is transferred from Tyr-tRNATyr to an active serine (S88) was also suggested for Rv2275, using radioactive labelling (12). Moreover, the S88 residue of Rv2275 was suggested to be activated by the conserved Y253 residue, which corresponds to Y202 in AlbC (12). To verify the existence of a covalent intermediate, we further analysed AlbC and the five variants S37A, S37C, Y178F, E182Q and Y202F. The enzymes were incubated in the presence of [3H]Phe-tRNAPhe, and they were separated by SDS–PAGE before being transferred on a PVDF membrane, which was analysed with a radioimager. As observed for Rv2275, wild-type AlbC retained a high amount of the radiolabel, whereas the variant S37A did not retain any radiolabel. The variant S37C presented behaviour similar to that of the wild-type enzyme, confirming the formation of a covalent intermediate and the probable status of S37 as the point of covalent attachment (Figure 6). The variants Y178F and E182Q retained significant amounts of the radiolabel although much less than the wild-type enzyme (Figure 6). This result, combined with the substantial and complete loss of enzyme activity for the variants Y178F and E182Q, respectively (Figure 5D), argues that Y178 and E182 may play a crucial role in the good positioning of the aminoacyl moiety of the substrate in the active site—essential for an efficient formation of the covalent intermediate—concerning the variant Y202F, it retained ∼11% of the enzymatic activity (Figure 5D), and at least as much radiolabel as the wild-type enzyme (Figure 6). As the removal of the hydroxyl group in the variant Y202F does not prevent the formation of the covalent intermediate, this demonstrated that Y202 is not responsible for the S37 activation in AlbC.Figure 6.

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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