Chemical Rescue of Active Site Mutants of S. pneumoniae Surface Endonuclease EndA and Other Nucleases of the HNH Family by Imidazole

Abstract

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response.

Structural characterization of the virulence factor nuclease A fromStreptococcus agalactiae

“…Because overexpression of wild-type GBS_NucA in E. coli is difficult owing to toxicity (Derré -Bobillot et al, 2013), all biochemical analyses of surface mutations was performed on the background of a catalytically deactivated mutant, the activity of which could be re-introduced using an imidazole chemical rescue technique (Midon et al, 2012;Moon et al, 2011). This technique is founded on the basic principle that exogenously added imidazole functions as an effective mimic of the absent histidine thought to serve as the general base (Lehoux & Mitra, 1999).…”

“…It should be noted that the conditions for the imidazole rescue assay were optimized to produce maximal GBS_NucA (H148G) activity, and that the final conditions differ drastically from those used for EndA (H160G) (Midon et al, 2012;Moon et al, 2011). The pH optimum for GBS_NucA is considerably lower than for EndA (pH 6.5 versus pH 8, respectively) and the divalent-ion concentration is two orders of magnitude lower (0.1 mM versus 10 mM for EndA).…”

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

“…A well conserved histidine (H545 in NColE7) is responsible for generating the nucleophilic hydroxide by deprotonating a water molecule (Supporting Information Fig. S1) . The general acid residue that protonates the leaving group was not yet identified.…”

Inhibitors of Streptococcus pneumoniae Surface Endonuclease EndA Discovered by High-Throughput Screening Using a PicoGreen Fluorescence Assay

“…17 We therefore elected to use EndA(H160G) for assay development, and imidazole can be considered a cofactor in assays measuring the imidazole-dependent nuclease activity of EndA(H160G). 26 …”