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Structure of the archaeal pab87 peptidase reveals a novel self-compartmentalizing protease family.

Delfosse V, Girard E, Birck C, Delmarcelle M, Delarue M, Poch O, Schultz P, Mayer C - PLoS ONE (2009)

Bottom Line: A 20 A wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 A wide central chamber holding the eight active sites.Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism.We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche des Cordeliers, LRMA, INSERM UMR-S 872, Université Pierre et Marie Curie, Paris, France.

ABSTRACT
Self-compartmentalizing proteases orchestrate protein turnover through an original architecture characterized by a central catalytic chamber. Here we report the first structure of an archaeal member of a new self-compartmentalizing protease family forming a cubic-shaped octamer with D(4) symmetry and referred to as CubicO. We solved the structure of the Pyrococcus abyssi Pab87 protein at 2.2 A resolution using the anomalous signal of the high-phasing-power lanthanide derivative Lu-HPDO3A. A 20 A wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 A wide central chamber holding the eight active sites. Surprisingly, activity assays revealed that Pab87 degrades specifically d-amino acid containing peptides, which have never been observed in archaea. Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism. We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

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Exopeptidase activities of the CubicO proteases.A, MS analysis of the products of the carboxypeptidation reaction catalyzed by Pab87. Peaks at m/z 1108.5 and 1037.5 correspond to the GM-penta-(Ala-Ala) substrate and to the GM-tetra-(Ala-Ala) product respectively. B, Effect of temperature on the Pab87 catalytic efficiency on d-Ala-p-nitroanilide. Box indicates the relative aminopeptidase activity at 90°C of Pab87 on l- and d-p-NA. C, Close-up view of the superimposed active sites of Pab87 (in cyan), O. anthropi DAP d-aminopeptidase (in white; PDB id 1EI5) and of Streptomyces R61 dd-carboxypeptidase (in green; PDB id 3PTE). Only side chains of catalytic residues of the three structures with backbone of Pab87 are represented. Secondary structure elements are indicated in bold.
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pone-0004712-g003: Exopeptidase activities of the CubicO proteases.A, MS analysis of the products of the carboxypeptidation reaction catalyzed by Pab87. Peaks at m/z 1108.5 and 1037.5 correspond to the GM-penta-(Ala-Ala) substrate and to the GM-tetra-(Ala-Ala) product respectively. B, Effect of temperature on the Pab87 catalytic efficiency on d-Ala-p-nitroanilide. Box indicates the relative aminopeptidase activity at 90°C of Pab87 on l- and d-p-NA. C, Close-up view of the superimposed active sites of Pab87 (in cyan), O. anthropi DAP d-aminopeptidase (in white; PDB id 1EI5) and of Streptomyces R61 dd-carboxypeptidase (in green; PDB id 3PTE). Only side chains of catalytic residues of the three structures with backbone of Pab87 are represented. Secondary structure elements are indicated in bold.

Mentions: As Pab87 is a S12 protease, the amino and carboxypeptidase activities were investigated. The aminopeptidase activity was tested using l- and d-Ala-p-nitroanilide compounds as substrates. Preliminary studies underlined the high Km values for these substrates. Catalytic efficiency kcat/Km could be determined as a function of temperature (Figure 3B). Clearly, the enzyme shows a preference for the substrate with a d configuration. At 90°C, the kcat/Km is 3.92 M−1 s−1 and 0.06 M−1 s−1 for respectively the d- and l-Ala-p-nitroanilide. The kcat/Km values increase drastically after 70°C. The maximum of activity was obtained at 90°C, the maximum technically reachable temperature, underlying the hyperthermophilic properties of the enzyme. Moreover, dd-carboxypeptidase activity was observed on various muropeptides. Mass spectrometry analyzes clearly show that Pab87 is able to hydrolyse the C-terminal d-alanine of pentapeptidic precursors with or without the sugar moiety from both Gram-negative and Gram-positive bacteria, at 37 and 90°C (Figure 3A). The d-stereospecificity observed for Pab87 is consistent with the activities of the majority of S12 proteases. Catalytic residues of P. abyssi CubicO protease perfectly superimpose to those of other S12 members, indicating similar catalytic pathways despite different subtrate specificities (Figure 3C). In conclusion, Pab87 is a hyperthermophilic peptidase that acts on d-amino acid containing peptides at N- and C-termini.


Structure of the archaeal pab87 peptidase reveals a novel self-compartmentalizing protease family.

Delfosse V, Girard E, Birck C, Delmarcelle M, Delarue M, Poch O, Schultz P, Mayer C - PLoS ONE (2009)

Exopeptidase activities of the CubicO proteases.A, MS analysis of the products of the carboxypeptidation reaction catalyzed by Pab87. Peaks at m/z 1108.5 and 1037.5 correspond to the GM-penta-(Ala-Ala) substrate and to the GM-tetra-(Ala-Ala) product respectively. B, Effect of temperature on the Pab87 catalytic efficiency on d-Ala-p-nitroanilide. Box indicates the relative aminopeptidase activity at 90°C of Pab87 on l- and d-p-NA. C, Close-up view of the superimposed active sites of Pab87 (in cyan), O. anthropi DAP d-aminopeptidase (in white; PDB id 1EI5) and of Streptomyces R61 dd-carboxypeptidase (in green; PDB id 3PTE). Only side chains of catalytic residues of the three structures with backbone of Pab87 are represented. Secondary structure elements are indicated in bold.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651629&req=5

pone-0004712-g003: Exopeptidase activities of the CubicO proteases.A, MS analysis of the products of the carboxypeptidation reaction catalyzed by Pab87. Peaks at m/z 1108.5 and 1037.5 correspond to the GM-penta-(Ala-Ala) substrate and to the GM-tetra-(Ala-Ala) product respectively. B, Effect of temperature on the Pab87 catalytic efficiency on d-Ala-p-nitroanilide. Box indicates the relative aminopeptidase activity at 90°C of Pab87 on l- and d-p-NA. C, Close-up view of the superimposed active sites of Pab87 (in cyan), O. anthropi DAP d-aminopeptidase (in white; PDB id 1EI5) and of Streptomyces R61 dd-carboxypeptidase (in green; PDB id 3PTE). Only side chains of catalytic residues of the three structures with backbone of Pab87 are represented. Secondary structure elements are indicated in bold.
Mentions: As Pab87 is a S12 protease, the amino and carboxypeptidase activities were investigated. The aminopeptidase activity was tested using l- and d-Ala-p-nitroanilide compounds as substrates. Preliminary studies underlined the high Km values for these substrates. Catalytic efficiency kcat/Km could be determined as a function of temperature (Figure 3B). Clearly, the enzyme shows a preference for the substrate with a d configuration. At 90°C, the kcat/Km is 3.92 M−1 s−1 and 0.06 M−1 s−1 for respectively the d- and l-Ala-p-nitroanilide. The kcat/Km values increase drastically after 70°C. The maximum of activity was obtained at 90°C, the maximum technically reachable temperature, underlying the hyperthermophilic properties of the enzyme. Moreover, dd-carboxypeptidase activity was observed on various muropeptides. Mass spectrometry analyzes clearly show that Pab87 is able to hydrolyse the C-terminal d-alanine of pentapeptidic precursors with or without the sugar moiety from both Gram-negative and Gram-positive bacteria, at 37 and 90°C (Figure 3A). The d-stereospecificity observed for Pab87 is consistent with the activities of the majority of S12 proteases. Catalytic residues of P. abyssi CubicO protease perfectly superimpose to those of other S12 members, indicating similar catalytic pathways despite different subtrate specificities (Figure 3C). In conclusion, Pab87 is a hyperthermophilic peptidase that acts on d-amino acid containing peptides at N- and C-termini.

Bottom Line: A 20 A wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 A wide central chamber holding the eight active sites.Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism.We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche des Cordeliers, LRMA, INSERM UMR-S 872, Université Pierre et Marie Curie, Paris, France.

ABSTRACT
Self-compartmentalizing proteases orchestrate protein turnover through an original architecture characterized by a central catalytic chamber. Here we report the first structure of an archaeal member of a new self-compartmentalizing protease family forming a cubic-shaped octamer with D(4) symmetry and referred to as CubicO. We solved the structure of the Pyrococcus abyssi Pab87 protein at 2.2 A resolution using the anomalous signal of the high-phasing-power lanthanide derivative Lu-HPDO3A. A 20 A wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 A wide central chamber holding the eight active sites. Surprisingly, activity assays revealed that Pab87 degrades specifically d-amino acid containing peptides, which have never been observed in archaea. Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism. We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

Show MeSH
Related in: MedlinePlus