| Abstract: |
Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5′-OH termini of DNA or RNA polynucleotides and Ni2+/Mn2+-dependent dephosphorylation of 2′,3′ cyclic phosphate, 2′-phosphate, and 3′-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni2+-dependent phosphatase activity with 2′-AMP and 3′-AMP. Eight of the 11 CthPnkp side chains were also required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage λ phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2′-AMP or 3′-AMP were dispensable for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424). Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 RNA Society. |
