Structure-function analysis of the phosphoesterase component of the nucleic acid end-healing enzyme Runella slithyformis HD-Pnk Journal Article
| Authors: | Munir, A.; Shuman, S. |
| Article Title: | Structure-function analysis of the phosphoesterase component of the nucleic acid end-healing enzyme Runella slithyformis HD-Pnk |
| Abstract: | Runella slithyformis HD-Pnk is the prototype of a family of dual 5' and 3' nucleic acid end-healing enzymes that phosphorylate 5'-OH termini and dephosphorylate 2',3'-cyclic-PO4, 3'-PO4, and 2'-PO4 ends. HD-Pnk is composed of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Here, we probed the phosphoesterase activity of HD-Pnk by querying its ability to hydrolyze non-nucleic acid phosphoester substrates and by conducting a mutational analysis of conserved amino acid constituents of the HD domain. We report that HD-Pnk catalyzes vigorous hydrolysis of p-nitrophenylphosphate (Km = 3.13 mM; kcat = 27.8 s-1) using copper as its metal cofactor. Mutagenesis identified Gln28, His33, His73, Asp74, Lys77, His94, His127, Asp162, and Arg166 as essential for p-nitrophenylphosphatase and DNA 3' phosphatase activities. Structural modeling places these residues at the active site, wherein His33, His73, Asp74, His94, and His127 are predicted to coordinate a binuclear metal complex and Lys77 and Arg166 engage the scissile phosphate. HD-Pnk homologs are distributed broadly (and exclusively) in bacteria, usually in a two-gene cluster with a putative ATP-dependent polynucleotide ligase (LIG). We speculate that HD-Pnk and LIG comprise the end-healing and end-sealing components of a bacterial nucleic acid repair pathway.IMPORTANCE 5'-end healing and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH ends are phosphorylated by a polynucleotide kinase, and 3'-PO4 or 2',3'-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate 5'-PO4 and 3'-OH termini needed for joining by DNA and RNA ligases. This study interrogates, biochemically and via mutagenesis, the phosphoesterase activity of Runella slithyformis HD-Pnk, a bifunctional bacterial 5'- and 3'-end-healing enzyme composed of HD phosphoesterase and P-loop kinase modules. HD-Pnk homologs are found in 129 bacterial genera from 11 phyla. In 123/129 instances, HD-Pnk is encoded in an operon-like gene cluster with a putative ATP-dependent polynucleotide ligase (LIG), suggesting that HD-Pnk and LIG are agents of a conserved bacterial nucleic acid repair pathway. Copyright © 2019 American Society for Microbiology. |
| Keywords: | genetics; protein domain; metabolism; enzymology; bacterial protein; chemistry; bacterial proteins; amino acid sequence; sequence alignment; bacterial dna; catalytic domain; dna, bacterial; copper; polynucleotide 5' hydroxyl kinase; polynucleotide 5'-hydroxyl-kinase; enzyme active site; protein domains; polynucleotide kinase; operon; hd domain; nucleic acid repair; flexibacteraceae; cytophagaceae; 3′ phosphatase; 4 nitrophenylphosphatase; 4-nitrophenylphosphatase |
| Journal Title: | Journal of Bacteriology |
| Volume: | 201 |
| Issue: | 16 |
| ISSN: | 0021-9193 |
| Publisher: | American Society for Microbiology |
| Date Published: | 2019-08-01 |
| Start Page: | e00292-19 |
| Language: | English |
| DOI: | 10.1128/jb.00292-19 |
| PUBMED: | 31160396 |
| PROVIDER: | scopus |
| PMCID: | PMC6657592 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 July 2020 -- Source: Scopus |
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