129 research outputs found
Shedding X-ray Light on the Role of Magnesium in the Activity of Mycobacterium tuberculosis Salicylate Synthase (MbtI) for Drug Design
The Mg2+-dependent Mycobacterium tuberculosis salicylate synthase (MbtI) is a key enzyme involved in the biosynthesis of siderophores. Because iron is essential for the survival and pathogenicity of the microorganism, this protein constitutes an attractive target for antitubercular therapy, also considering the absence of homologous enzymes in mammals. An extension of the structure-activity relationships of our furan-based candidates allowed us to disclose the most potent competitive inhibitor known to date (10, Ki = 4 ÎŒM), which also proved effective on mycobacterial cultures. By structural studies, we characterized its unexpected Mg2+-independent binding mode. We also investigated the role of the Mg2+ cofactor in catalysis, analyzing the first crystal structure of the MbtI-Mg2+-salicylate ternary complex. Overall, these results pave the way for the development of novel antituberculars through the rational design of improved MbtI inhibitors
CNTN6 mutations are risk factors for abnormal auditory sensory perception in autism spectrum disorders
Contactin genes CNTN5 and CNTN6 code for neuronal cell adhesion molecules that promote neurite outgrowth in sensory-motor neuronal pathways. Mutations of CNTN5 and CNTN6 have previously been reported in individuals with autism spectrum disorders (ASDs), but very little is known on their prevalence and clinical impact. In this study, we identified CNTN5 and CNTN6 deleterious variants in individuals with ASD. Among the carriers, a girl with ASD and attention-deficit/hyperactivity disorder was carrying five copies of CNTN5. For CNTN6, both deletions (6/1534 ASD vs 1/8936 controls; P=0.00006) and private coding sequence variants (18/501 ASD vs 535/33480 controls; P=0.0005) were enriched in individuals with ASD. Among the rare CNTN6 variants, two deletions were transmitted by fathers diagnosed with ASD, one stop mutation CNTN6W923X was transmitted by a mother to her two sons with ASD and one variant CNTN6P770L was found de novo in a boy with ASD. Clinical investigations of the patients carrying CNTN5 or CNTN6 variants showed that they were hypersensitive to sounds (a condition called hyperacusis) and displayed changes in wave latency within the auditory pathway. These results reinforce the hypothesis of abnormal neuronal connectivity in the pathophysiology of ASD and shed new light on the genes that increase risk for abnormal sensory perception in ASD
Biological and structural characterization of theMycobacterium smegmatis nitroreductase NfnB, and its rolein benzothiazinone resistance
Tuberculosis is still a leading cause of death in developing
countries, for which there is an urgent need
for new pharmacological agents. The synthesis of
the novel antimycobacterial drug class of benzothiazinones
(BTZs) and the identification of their
cellular target as DprE1 (Rv3790), a component of
the decaprenylphosphoryl-b-D-ribose 2'-epimerase
complex, have been reported recently. Here, we
describe the identification and characterization of a
novel resistance mechanism to BTZ in Mycobacterium
smegmatis. The overexpression of the nitroreductase
NfnB leads to the inactivation of the drug by
reduction of a critical nitro-group to an amino-group.
The direct involvement of NfnB in the inactivation of
the lead compound BTZ043 was demonstrated by
enzymology, microbiological assays and gene knockout
experiments. We also report the crystal structure
of NfnB in complex with the essential cofactor flavin
mononucleotide, and show that a common amino
acid stretch between NfnB and DprE1 is likely to be
essential for the interaction with BTZ. We performed
docking analysis of NfnB-BTZ in order to understand
their interaction and the mechanism of
nitroreduction. Although Mycobacterium tuberculosis
seems to lack nitroreductases able to inactivate
these drugs, our findings are valuable for the design
of new BTZ molecules, which may be more effective
in vivo
The bacterial stressosome:a modular system that has been adapted to control secondary messenger signaling
SummaryThe stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor ÏB, leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome
Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses
The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny
Techniques and Applications: The heterologous expression of Mycobacterium tuberculosis genes is an uphill road.
In the field of tuberculosis, the challenge is to interpret genome information in the context of the hostâpathogen interaction, and then translate the resulting knowledge into a new generation of drugs and vaccines with which to arm ourselves in the fight against this ancient disease.
Structural genomics, the large-scale determination of protein structures, promises to provide many useful data for drug discovery. To date, 38 protein structures have been determined from Mycobacterium tuberculosis. Most are those of proteins involved in intermediary metabolism and lipid metabolism. These structures will provide the opportunity to undertake structure-guided drug discovery programs in the search for new classes of antibiotics.
Existing information will provide spin-offs for future applications, but the way ahead is still arduous because it is difficult to obtain sufficient yields of the functionally active gene products, despite numerous efforts to optimize the prokaryotic expression systems. More protein candidates, responsible for virulence, intracellular survival, transcriptional regulation or drug resistance, must be characterized to develop new treatments for tuberculosis
A dual conformation of the post-decarboxylation intermediate is associated with distinct enzyme states in mycobacterial KGD (α-ketoglutarate decarboxylase)
International audienceα-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of α-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by α-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain α-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the α-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (α-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o
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