Chemical rescue of active site mutants of S. pneumoniae surface endonuclease EndA and other nucleases of the HNH family by imidazole

Chembiochem : a European Journal of Chemical Biology
Marika MidonAlfred Pingoud

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)....Continue Reading

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Citations

Mar 13, 2016·Nucleic Acids Research·Andrea F MoonLars C Pedersen
Jul 16, 2013·Letters in Applied Microbiology·N S JakubovicsJ G Burgess
Jun 5, 2014·Protein Science : a Publication of the Protein Society·Eszter NémethBéla Gyurcsik
Nov 6, 2014·Acta Crystallographica. Section D, Biological Crystallography·Andrea F MoonLars C Pedersen
Aug 23, 2018·Frontiers in Cellular and Infection Microbiology·Ya-Jie LiYue-Zhong Li

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