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.