Crystal structure of E. coli endonuclease V, an essential enzyme for deamination repair

Abstract

Endonuclease V (EndoV) is a ubiquitous protein present in all three kingdoms of life, responsible for the specific cleavages at the second phosphodiester bond 3’ to inosine. E. coli EndoV (EcEndoV) is the first member discovered in the EndoV family. It is a small protein with a compact gene organization, yet with a wide spectrum of substrate specificities. However, the structural basis of its substrate recognition is not well understood. In this study, we determined the 2.4 Å crystal structure of EcEndoV. The enzyme preserves the general ‘RNase H-like motif’ structure. Two subunits are almost fully resolved in the asymmetric unit, but they are not related by any 2-fold axes. Rather, they establish “head-to-shoulder” contacts with loose interactions between each other. Mutational studies show that mutations that disrupt the association mode of the two subunits also decrease the cleavage efficiencies of the enzyme. Further biochemical studies suggest that EcEndoV is able to bind to single-stranded, undamaged DNA substrates without sequence specificity, and forms two types of complexes in a metal-independent manner, which may explain the wide spectrum of substrate specificities of EcEndoV.

Crystal structure and MD simulation of mouse EndoV reveal wedge motif plasticity in this inosine-specific endonuclease

“…4b,c ). The wedge motifs in hEndoV and also EcEndoV (PDB entry 4xpu) 27 are retracted about 4–6 Å compared with the DNA bound TmEndoV, but the conserved Tyr91 in hEndoV 26 is still pointing towards the DNA/RNA binding groove, with side chain torsion angle χ 1 being ~−170° ( trans ) ( Fig. 4d ).…”

“…A series of prokaryotic and eukaryotic EndoV homologs have recently been studied with respect to catalysis, substrate specificity and structure 12 21 26 27 28 . The human homolog has activity for inosines in RNA, while the bacterial versions can cleave both RNA and DNA with inosines.…”

A restatement of recent advances in the natural science evidence base concerning neonicotinoid insecticides and insect pollinators

“…Models of the effects of sublethal stress, including insecticide exposure, on bumblebee colony dynamics and failure rates have also been developed. [E xp_op ] A29 Budge et al [ 13 ] collected data on honeybee colony in-season loss and neonicotinoid use from nine regions of the UK every other year from 2000 to 2010. Controlling for region (but not year) they find a weak but significant correlation between colony loss and imidacloprid use, but not total neonicotinoid use.…”

Structural and biochemical insights into the 2′‐O‐methylation of pyrimidines 34 in tRNA

“…The EMSA experiments followed a previous protocol [37]. The standard 10 lL binding reaction mixture contained 40 mM Tris/HCl (pH 8.0), 20 mM KCl, 4 mM MgCl 2 , 2 mM DTT, 5% glycerol, 1 lM in vitro-transcribed TttRNA Leu CAA and various amounts of TtTrmL.…”