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Structure and mechanism of the 2',3' phosphatase component of the bacterial Pnkp-Hen1 RNA repair system.

Wang LK, Smith P, Shuman S - Nucleic Acids Res. (2013)

Bottom Line: The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376.A continuous tract of positive surface potential flanking the active site suggests an RNA binding site.The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

ABSTRACT
Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5' kinase, a central 2',3' phosphatase and a C-terminal ligase. The phosphatase module is a Mn(2+)-dependent phosphodiesterase-monoesterase that dephosphorylates 2',3'-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn(2+) and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn(2+) in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.

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The Pnkp-Hen1 RNA repair pathway. (A) The adjacent co-oriented ORFs encoding the Hen1 and Pnkp polypeptides comprise a bacterial RNA repair cassette conserved in taxa from 10 different bacterial phyla. Pnkp is a trifunctional RNA repair enzyme composed of 5′-OH polynucleotide kinase, 2′,3′ phosphatase and ligase domains. Hen1 is composed of a C-terminal methyltransferase catalytic domain (MTase) fused to a unique N-terminal module that binds to the Pnkp ligase domain. (B) 2′,3′-cyclic phosphate and 5′-OH ends are substrates for healing and sealing by Pnkp and Hen1. The 5′ end is phosphorylated by the Pnkp kinase, and the 2′,3′-cyclic phosphate is removed by the Pnkp phosphatase. The Hen1 methyltransferase installs a 2′-OCH3 mark at the terminal ribonucleoside before ligation of the ends. The ligation reaction is mediated by a heterodimer of the Pnkp ligase domain and the Hen1 N-terminal domain. The repair junction with the methyl mark is then resistant to scission by transesterifying endoribonucleases.
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gkt221-F1: The Pnkp-Hen1 RNA repair pathway. (A) The adjacent co-oriented ORFs encoding the Hen1 and Pnkp polypeptides comprise a bacterial RNA repair cassette conserved in taxa from 10 different bacterial phyla. Pnkp is a trifunctional RNA repair enzyme composed of 5′-OH polynucleotide kinase, 2′,3′ phosphatase and ligase domains. Hen1 is composed of a C-terminal methyltransferase catalytic domain (MTase) fused to a unique N-terminal module that binds to the Pnkp ligase domain. (B) 2′,3′-cyclic phosphate and 5′-OH ends are substrates for healing and sealing by Pnkp and Hen1. The 5′ end is phosphorylated by the Pnkp kinase, and the 2′,3′-cyclic phosphate is removed by the Pnkp phosphatase. The Hen1 methyltransferase installs a 2′-OCH3 mark at the terminal ribonucleoside before ligation of the ends. The ligation reaction is mediated by a heterodimer of the Pnkp ligase domain and the Hen1 N-terminal domain. The repair junction with the methyl mark is then resistant to scission by transesterifying endoribonucleases.

Mentions: The Pnkp-Hen1 RNA repair system of Clostridium thermocellum (Cth) is encoded in an operon-like gene cassette (Figure 1A). Pnkp-Hen1 systems are present in diverse bacteria from 10 different phyla. CthPnkp is the end-healing and end-sealing component and is composed of three catalytic domains: N-terminal kinase, central phosphatase and C-terminal ligase (2). The kinase module catalyzes phosphoryl transfer from ATP to the 5′-OH RNA end. The phosphatase domain releases Pi from 2′-PO4, 3′-PO4 or 2′,3′-cyclic phosphate ribonucleotides (Figure 1B). The ligase domain reacts with ATP to form a covalent enzyme–AMP adduct, just as classic RNA ligases do during strand joining, but it is unable per se to seal RNA strands (2,3). The sealing function of the Pnkp ligase domain is activated by the N-terminal half of the Hen1 protein (4). The C-terminal half of Hen1 is an autonomous Mn2+-dependent 3′-terminal ribose 2′-O-methyltransferase that installs a 2′-OCH3 mark at the RNA repair junction before ligation, and thereby immunizes the sealed RNA against recurrent endonuclease cleavage (Figure 1B) (5–8).Figure 1.


Structure and mechanism of the 2',3' phosphatase component of the bacterial Pnkp-Hen1 RNA repair system.

Wang LK, Smith P, Shuman S - Nucleic Acids Res. (2013)

The Pnkp-Hen1 RNA repair pathway. (A) The adjacent co-oriented ORFs encoding the Hen1 and Pnkp polypeptides comprise a bacterial RNA repair cassette conserved in taxa from 10 different bacterial phyla. Pnkp is a trifunctional RNA repair enzyme composed of 5′-OH polynucleotide kinase, 2′,3′ phosphatase and ligase domains. Hen1 is composed of a C-terminal methyltransferase catalytic domain (MTase) fused to a unique N-terminal module that binds to the Pnkp ligase domain. (B) 2′,3′-cyclic phosphate and 5′-OH ends are substrates for healing and sealing by Pnkp and Hen1. The 5′ end is phosphorylated by the Pnkp kinase, and the 2′,3′-cyclic phosphate is removed by the Pnkp phosphatase. The Hen1 methyltransferase installs a 2′-OCH3 mark at the terminal ribonucleoside before ligation of the ends. The ligation reaction is mediated by a heterodimer of the Pnkp ligase domain and the Hen1 N-terminal domain. The repair junction with the methyl mark is then resistant to scission by transesterifying endoribonucleases.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675462&req=5

gkt221-F1: The Pnkp-Hen1 RNA repair pathway. (A) The adjacent co-oriented ORFs encoding the Hen1 and Pnkp polypeptides comprise a bacterial RNA repair cassette conserved in taxa from 10 different bacterial phyla. Pnkp is a trifunctional RNA repair enzyme composed of 5′-OH polynucleotide kinase, 2′,3′ phosphatase and ligase domains. Hen1 is composed of a C-terminal methyltransferase catalytic domain (MTase) fused to a unique N-terminal module that binds to the Pnkp ligase domain. (B) 2′,3′-cyclic phosphate and 5′-OH ends are substrates for healing and sealing by Pnkp and Hen1. The 5′ end is phosphorylated by the Pnkp kinase, and the 2′,3′-cyclic phosphate is removed by the Pnkp phosphatase. The Hen1 methyltransferase installs a 2′-OCH3 mark at the terminal ribonucleoside before ligation of the ends. The ligation reaction is mediated by a heterodimer of the Pnkp ligase domain and the Hen1 N-terminal domain. The repair junction with the methyl mark is then resistant to scission by transesterifying endoribonucleases.
Mentions: The Pnkp-Hen1 RNA repair system of Clostridium thermocellum (Cth) is encoded in an operon-like gene cassette (Figure 1A). Pnkp-Hen1 systems are present in diverse bacteria from 10 different phyla. CthPnkp is the end-healing and end-sealing component and is composed of three catalytic domains: N-terminal kinase, central phosphatase and C-terminal ligase (2). The kinase module catalyzes phosphoryl transfer from ATP to the 5′-OH RNA end. The phosphatase domain releases Pi from 2′-PO4, 3′-PO4 or 2′,3′-cyclic phosphate ribonucleotides (Figure 1B). The ligase domain reacts with ATP to form a covalent enzyme–AMP adduct, just as classic RNA ligases do during strand joining, but it is unable per se to seal RNA strands (2,3). The sealing function of the Pnkp ligase domain is activated by the N-terminal half of the Hen1 protein (4). The C-terminal half of Hen1 is an autonomous Mn2+-dependent 3′-terminal ribose 2′-O-methyltransferase that installs a 2′-OCH3 mark at the RNA repair junction before ligation, and thereby immunizes the sealed RNA against recurrent endonuclease cleavage (Figure 1B) (5–8).Figure 1.

Bottom Line: The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376.A continuous tract of positive surface potential flanking the active site suggests an RNA binding site.The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

ABSTRACT
Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5' kinase, a central 2',3' phosphatase and a C-terminal ligase. The phosphatase module is a Mn(2+)-dependent phosphodiesterase-monoesterase that dephosphorylates 2',3'-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn(2+) and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn(2+) in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.

Show MeSH
Related in: MedlinePlus