Computational and mutational analysis of TatD DNase of Bacillus anthracis

Abstract

The role of TatD DNases as DNA repair enzymes or cell death (apoptotic) nucleases is well established in prokaryotes as well as eukaryotes. The current study aims to characterize the TatD nuclease from Bacillus anthracis (Ba TatD) and to explore its key histidine catalytic residues. Ba TatD was found to be a metal-dependent, nonspecific endonuclease which could efficiently cleave double-stranded DNA substrates. Moreover, Ba TatD nuclease was observed to be thermostable up to 55°C and act in a wide pH range indicating its industrial applicability. Diethyl pyrocarbonate-based histidine-selective alkylation of the Ba TatD resulted in a loss of its nuclease activity suggesting a crucial role of the histidine residues in its activity. The key residues of Ba TatD were predicted using sequence analysis and structure-based approaches, and then the predicted residues were further tested by mutational analysis. Upon mutational analysis, H128 and H153 have been found to be crucial for Ba TatD activity, though H153 seems to bear an important but a dispensable role for the Ba TatD nuclease. Ba TatD had a uniform expression in the cytosol of B. anthracis, which indicates a significant role of the protein in the pathogen's life cycle. This is the first study to identify and characterize the TatD DNase from B. anthracis and will be helpful in gaining more insights on the role of TatD proteins in Gram-positive bacteria where it remains unexplored. K E Y W O R D S active site, Bacillus anthracis, histidine, TatD nuclease, thermostable

A structural study of TatD from Staphylococcus aureus elucidates a putative DNA-binding mode of a Mg2+-dependent nuclease

“…In addition, E. coli TatD only showed nuclease activity for single-stranded nucleic acids, while SaTatD efficiently trimmed dsDNA. This functional difference may be caused by the distinct difference in catalytic residues and may be owing to the evolutionary selection for a specific activity within the ubiquitous TIM-barrel fold (Singh et al, 2019).…”

Human and bacterial TatD enzymes exhibit apurinic/apyrimidinic (AP) endonuclease activity

“…Several prokaryotic and eukaryotic TatD proteins have been characterized as deoxyribonucleases in general, and in most cases as 3′-exonucleases, with varying substrate specificities ( 33 , 35–37 , 39 , 40 , 64 , 65 ). We tested the nuclease activities of human TATDN1 and TATDN3 on short (25-mer) oligodeoxynucleotide substrates to assess exonuclease polarity and preference for single-stranded (ss) versus double-stranded (ds) DNA.…”

“…TatD proteins have been characterized only as Mg 2+ -dependent nucleases ( 33 , 35–37 , 39 , 40 , 64 , 65 ), although Mg 2+ , Zn 2+ , Mn 2+ and Ni 2+ have been observed in the active sites of several TatD structures in the PDB ( Supplementary Table S3 ). Based on this and the modulation of AP endo and 3′-exo activities by Ca 2+ , we characterized the effects of Zn 2+ , Mn 2+ and Ni 2+ on the AP endo activities of TATDNs.…”

Investigating the pathogenic SNPs in BLM helicase and their biological consequences by computational approach

“…The application of computational biology in genome research is well established 18 , 72 and is frequently used to filter for the most potential deleterious nsSNPs in target genes to understand the aetiology of various diseases 28 , 73 – 75 . Computational analysis can provide molecular insight into the changes in protein structure and function due to point mutations in the target genes.…”