Enzyme–adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface

Abstract

DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

Analysis of the distribution and evolution of the ATP‐dependent DNA ligases of bacteria delineates a distinct phylogenetic group ‘Lig E’

“…The ligase domain of the Lig D1 enzyme from the human pathogen M. tuberculosis (PDB 1VS0) was crystallized without its additional POL or PE domains (Akey et al ., ), while more recently the Lig E b‐ADL, which lacks such domains, has been obtained from the marine psychrophile Psychromonas sp. strain SP041 (PDB 4D05) (Williamson et al ., ) (Fig. A).…”

“…A fourth group of ligases, which includes the first identified b‐ADL from H. influenzae (Cheng and Shuman, ) have activity with singly nicked DNA, and lower levels of joining with double‐stranded breaks, both cohesive and blunt ended, as well as mismatched and gapped substrates has also been detected (Cheng and Shuman, ; Magnet and Blanchard, ; Williamson et al ., ). These enzymes have only the AD and OB domains, and all have a predicted N‐terminal signal sequence that would direct the mature protein to the periplasmic space of the bacterium, concomitant with its proteolytic removal.…”

“…The present study investigates the structural organization of b‐ADLs and their distribution and diversity among bacterial taxa. Our analysis draws on the wealth of sequence information that has become available in recent years: a large number of bacterial genome sequences are now available, many b‐ADLs have been biochemically characterized (Weller et al ., ; Gong et al ., ; Magnet and Blanchard, ; Zhu and Shuman, 2005; 2007; de Vega, ; Williamson and Pedersen, ; Williamson et al ., ) and the structures of two b‐ADLs have been determined to high resolution (Akey et al ., ; Williamson et al ., ). We have first identified b‐ADLs in GenBank and classified them based on their domain arrangements.…”

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

“…Within a given taxon, two or more DNA ligase enzymes may coexist, often with a division of labor between them. Examples of minimal ATP-dependent ligases include bacteriophage T7 DNA ligase (3), Chlorella virus DNA ligase (4,5), bacterial nonhomologous end-joining (NHEJ) ligases LigD and LigC (6)(7)(8), and bacterial DNA ligase LigE (9,10). The more complex multidomain architectures of ATP-dependent DNA ligases are exemplified by mammalian enzymes Lig1, LigIII, and LigIV (11)(12)(13)(14).…”

Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore

“…32–36 However, the α-HL nanopore analysis is based on ion channel electrical measurements and requires significant electrolyte concentration to detect changes in the electrical current. 32, 37 In order to minimize the influence of monovalent cations, which perturb the activity of DNA ligase, polyethylene glycol (PEG) was added to the electrolyte to enhance the hydrophobic interactions between the ligase and DNA strands.…”