The human endonuclease III (hNTH1), a homolog of the <i>Escherichia coli</i> enzyme (Nth), is DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, full-length (304 amino acid residues) expressed as glutathione <i>S</i>-transferase fusion polypeptide <i>E. coli</i>. Purified wild-type protein two additional residues truncated deletion 22 at NH₂ terminus were equally active had...

The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as last resort to treat XDR-TB. Resistance the aminoglycoside kanamycin hallmark Here, we reveal function structure mycobacterial protein Eis responsible for resistance in significant fraction kanamycin-resistant Mycobacterium clinical isolates. We demonstrate that has an unprecedented ability acetylate multiple amines many...

Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while non-canonical depends on IKK1/α. structural and biochemical bases for distinct these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) X-ray crystal structures human IKK1 in dimeric (∼150 kDa) hexameric (∼450 forms. hexamer, which representative form but comprises only ∼2%...

Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activity-based metabolomic profiling method to assign function a unique phosphatase, Rv1692. In contrast its annotation as nucleotide kinetic characterization indicate that Rv1692 D,L- glycerol 3-phosphate phosphatase. Crystal structures reveal architecture, fusion predicted haloacid dehalogenase fold with...

The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing degenerate central region. Toward defining mechanistic principles affinity regulated degeneracy, we observed an unusual dependence the RelA on identity base pair, which appears be noncontacted in complex crystal structures. sites A or T at position is ~10-fold higher than G C. structures neither...

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits gamma-radiation resistance approaching that of Deinococcus radiodurans. The genome sequenced by U.S. Department Energy's Joint Genome Institute which suggested existence three replicons, 4.76 Mb linear chromosome, 0.18 plasmid, 12.92 Kb circular plasmid. Southern hybridization confirmed chromosome is linear. K. sequence examined to learn about physiology...

Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal replication. Biochemical assays measuring activity have been limited to monitoring formation of radioactively labelled because the intrinsically low catalytic efficiency DnaG. Furthermore, is prone aggregation and proteolytic degradation. These factors impeded discovery inhibitors by high-throughput screening (HTS). In this study, we expressed purified previously uncharacterized from Mycobacterium tuberculosis ( Mtb...

Abstract A recently discovered cause of tuberculosis resistance to a drug last resort, the aminoglycoside kanamycin, results from modification this by enhanced intracellular survival (Eis) protein. Eis is structurally and functionally unique acetyltransferase with an unusual capability acetylating aminoglycosides at multiple positions. The extent regioversatility its defining protein features are unclear. Herein, we determined positions order acetylation five NMR spectroscopy. This analysis...

Rapid and high-fidelity phosphorylation of two serines (S32 S36) IκBα by a prototype Ser/Thr kinase IKK2 is critical for fruitful canonical NF-κB activation. Here, we report that dual specificity autophosphorylates itself at tyrosine residues in addition to its activation loop serines. Mutation one such tyrosine, Y169, located proximity the active site, phenylalanine, renders inactive S32 IκBα. Surprisingly, auto-phosphorylated relayed phosphate group(s) without ATP when ADP present. We also...

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as monomer in removing the mutagenic adduct O6-alkylguanine (induced by alkylating carcinogens) via stoichiometric reaction. The alkyl group is transferred without cofactor to specific cysteine acceptor residue of MGMT, Cys-145 case human containing 207 amino acid residues and thereby inactivates protein. As prelude investigation reaction mechanism MGMT elucidation its structure free substrate-bound forms...

Novel antibiotics are needed to overcome the challenge of continually evolving bacterial resistance. This has led a renewed interest in mechanistic studies once popular like chloramphenicol (CAM). Chloramphenicol acetyltransferases (CATs) enzymes that covalently modify CAM, rendering it inactive against its target, ribosome, and thereby causing resistance CAM. Of three major types CAT (CAT(I-III)), CAM-specific CAT(III) been studied extensively. Much less is known about another clinically...

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon accessory ubiquitin (Ub)-binding essential modulator (NEMO)/IKKγ as well poly-Ub chains. However, mechanism which binding serves promote IKK unclear. Here, we show that NEMO/IKKγ linear promotes a second interaction between and IKK2/IKKβ, distinct from...

Hexameric DnaB helicase unwinds the DNA double helix during replication of genetic material in bacteria. is an essential bacterial protein; therefore, it important potential target for antibacterial drug discovery. We report a crystal structure N‐terminal region from pathogen Mycobacterium tuberculosis ( Mt DnaBn), determined at 2.0 Å resolution. This provides atomic resolution details formation hexameric ring by two distinct interfaces. An extensive hydrophobic interface stabilizes dimer...

DksA2 and bind by X‐ray crystallography (View interaction)

Abstract Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order functional, yet knowledge secondary structural features regulate exposure motifs across transcriptome not currently available. Using transcriptome-wide RNA information we show retained introns mouse commonly flanked a short (≲70 nucleotide), base-paired...

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, is responsible for the removal of wide variety alkylated base lesions in DNA, e.g., N-alkylpurines and cyclic ethenoadducts adenine, guanine, cytosine. These lesions, some which are mutagenic toxic, generated endogenously or by genotoxic agents such as N-alkylnitrosamines vinyl chloride. Wild-type mouse MPG, expressed from recombinant baculovirus, was purified to near homogeneity studying its specific interaction with...