Activity and structure of Pseudomonas putida MPE, a manganese-dependent single-strand DNA endonuclease encoded in a nucleic acid repair gene cluster

Anam Ejaz, Yehuda Goldgur, Stewart Shuman

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

A recently identified and widely prevalent prokaryal gene cluster encodes a suite of enzymes with imputed roles in nucleic acid repair. The enzymes are as follows: MPE, a DNA endonuclease; Lhr-Core, a 3'-5' DNA helicase; LIG, an ATP-dependent DNA ligase; and Exo, a metallo-β-lactamase-family nuclease. Bacterial and archaeal MPE proteins belong to the binuclear metallophosphoesterase superfamily that includes the wellstudiedDNArepair nucleases Mre11 and SbcD. Here, we report that the Pseudomonas putida MPE protein is a manganese-dependent DNA endonuclease that incises either linear single strands or the single-strand loops of stem-loop DNA structures. MPE has feeble activity on duplex DNA. A crystal structure of MPE at 2.2 A resolution revealed that the active site includes two octahedrally coordinated manganese ions. Seven signature amino acids of the binuclear metallophosphoesterase superfamily serve as the enzymic metal ligands in MPE: Asp33, His35, Asp78, Asn112, His124, His146, and His158. A swath of positive surface potential on either side of the active site pocket suggests a binding site for the single-strand DNA substrate. The structure of MPE differs from Mre11 and SbcD in several key respects: (i) MPE is a monomer, whereas Mre11 and SbcD are homodimers; (ii) MPE lacks the capping domain present in Mre11 and SbcD; and (iii) the topology of the β sandwich that comprises the core of the metallophosphoesterase fold differs in MPE vis-à-vis Mre11 and SbcD. We surmise that MPE exemplifies a novel clade of DNA endonuclease within the binuclear metallophosphoesterase superfamily.

Original languageEnglish
Pages (from-to)7931-7941
Number of pages11
JournalJournal of Biological Chemistry
Volume294
Issue number19
DOIs
StatePublished - 10 May 2019
Externally publishedYes

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