The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N -acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage λ to integrate or excise its genome into and out of the host chromosome. λ recombination is carried out by the bacteriophage-encoded integrase protein (λ -int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of λ -int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of λ -int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.λ -int catalyses an ordered, pair-wise exchange of four DNA strands between two different pairs of recombination substrates 1,2 . During integration, λ -int aligns the bacteriophage attachment site attP with the bacterial attachment site attB and recombines these sequences to generate the recombination joints attL and attR flanking the integrated prophage (Fig. 1a, b). During the transition to lytic growth, the bacteriophage DNA is excised to regenerate attP and attB. In both reactions, the analogous pair of DNA strands ('top' strands) is exchanged first 3,4 to form a branched, four-way DNA intermediate known as a Holliday junction. Subsequent exchange of 'bottom' strands resolves the Holliday junction into linear recombinant products 2 . Although integration and excision might appear to be reciprocal reactions ( Fig. 1), they involve different substrates and are effectively irreversible 5 . The recombination machinery is configured differently during integration or excision by two different overlapping subsets of accessory factors and binding sites that bend the DNA arms flanking the core sites of strand exchange 2,6 . DNA bending is a prerequisite for the simultaneous interactions with arm and core sites 6-9 that deliver λ -int to lower-affinity core sites 10 . Arm-binding interactions allosterically enhance the fidelity of DNA strand exchange 11 and bias the outcome of Holliday junction resolution in favour of the recombined products 12 .Correspondence and requests for materials should be addressed to T.E. (tome@hms.harvard.edu).. * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. These structures suggest that only a small shift in subunit packing is sufficient to redirect DNA cleavage and exchange activities from one pair of strands to the other, in order to resolve the Holliday junction into products 13,14 . However, the molecular mechanism o...

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH 2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 ؋ 10 4 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the K m was 47 nM, and k cat was ϳ0.6/min, independent of whether DHU paired with G or A. The enzyme carries out ␤-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptideoligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.Reactive oxygen species are generated as by-products of oxidative phosphorylation or by ionizing radiation and induce extensive base damage that is mainly repaired via the base excision repair (BER) 1 pathway. This repair is initiated by removal of the damaged base, catalyzed by a DNA glycosylase (1-3). There are two classes of DNA glycosylases with distinct substrate specificities: the monofunctional simple glycosylase and the glycosylase with associated AP lyase activity. All oxidized base lesions are removed from DNA by DNA glycosylase/AP lyases, which not only catalyze removal of the base lesion but also cause strand cleavage via ␤-elimination. The Escherichia coli endonuclease III (Nth) recognizes a wide range of oxidized pyrimidine derivatives, including ring-saturated and ring-fragmented derivatives such as thymine glycol (Tg), 5-hydroxycytosine, 5,6-dihydrouracil (DHU), and urea (1, 3-6). This enzyme has been well conserved from E. coli to the humans (7,8). On the other hand, oxidized purine lesions are also repaired by DNA glycosylase/AP lyases, i.e. Mut M (Fpg) of E. coli or OGG of eukaryotes (yeast and mammals), which do not share extensive sequence similarity. Furthermore, unlike these e...

No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through structure determination of four sequence-diverse M proteins in complex with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies targeting the M-C4BP interaction, and also inform a path towards vaccine design.

A recently discovered cause of resistance of tuberculosis to a drug of last resort, the aminoglycoside kanamycin, is modification of this drug by the enhanced intracellular survival (Eis) protein. Eis is a structurally and functionally unique acetyltransferase with an unusual capability of acetylating aminoglycosides at multiple positions. The extent of this regioversatility and its defining protein features are unclear. Herein, we determined the positions and order of acetylation of five aminoglycosides by NMR spectroscopy. This analysis revealed unprecedented acetylation of the 3"-amine of kanamycin, amikacin, and tobramycin, and γ-amine of the 4-amino-2-hydroxybutyryl group of amikacin. A crystal structure of Eis in complex with coenzyme A and tobramycin revealed how tobramycin can be accommodated in the Eis active site in two binding modes consistent with its di-acetylation. These studies describing chemical and structural details of acetylation will guide future efforts towards designing aminoglycosides and Eis inhibitors to overcome resistance in tuberculosis.

Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase–pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z′ = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery.

Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activitybased metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L-glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N-acetyltransferase region.Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis.haloacid dehalogenase superfamily | enzyme function | pathway discovery E ach year, 1.4 million people succumb to tuberculosis, making Mycobacterium tuberculosis the deadliest bacterium affecting mankind (1). In addition, the dissemination of strains resistant to several antibiotics underscores the need for better understanding of this pathogen and for the development of novel vaccines and therapeutics (2, 3). Our approach to elucidating the unique pervasiveness of M. tuberculosis is through comprehensive discovery and characterization of metabolic pathways, as metabolism underlies survival of the bacteria both inside and outside the host and can contribute to phenotypic and genetic drug resistance.The M. tuberculosis genome encodes for 4,043 genes, of which 3,933 encode proteins (4, 5). Many genes with essential functions, such as DNA replication, protein and RNA synthesis, and cell-division, have close homologs in other bacteria, and their functions are annotated largely on the basis of analysis of their counterparts. However, functions of at least one-third of the genes are unknown or putative (4). In particular, little is known about genes that are not conserved or conditionally important. Characterization of such genes presents a daunting task. M. tuberculosis is thought to be subjected to a myriad of conditions during its life cycle in the host, such as low pH, reactive oxygen and nitrogen species, and so on (6, 7). Understanding how M. tuberculosis adapts to and even thrives in such diverse environments is critical to our understanding of the pathology itself and for the discovery of novel therapeutics to treat tuberculosis.We applied an innovative, activity-based metabolomic profiling (ABMP) approach to assign function to orphan (without a priori known substrates/products) mycobacterial enzymes and to discover unk...

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6-alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spetroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.