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Nucleic Acids Res. 2007;35(22):7721-32. Epub 2007 Nov 5.

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase.

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1
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.

Abstract

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5' and 3' end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage lambda phosphatase (lambda-Pase). CthPnkp is a Ni(2+)/Mn(2+)-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn(2+)-dependent 2',3' cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2',3' cyclic phosphate to a 3',5' cyclic phosphate. A single H189D mutation imposes strict specificity for a 2',3' cyclic phosphate, which is cleaved to form a single 2'-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k(cat). We also characterize a previously unrecognized phosphodiesterase activity of lambda-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. lambda-Pase also has cyclic phosphodiesterase activity with nucleoside 2',3' cyclic phosphates, which it hydrolyzes to yield a mixture of 2'-NMP and 3'-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.

PMID:
17986465
PMCID:
PMC2190708
DOI:
10.1093/nar/gkm868
[Indexed for MEDLINE]
Free PMC Article
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