75 research outputs found
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 MBP fusion protein restores the activity of the first phosphatase domain of CD45.
CD45 is a receptor-like protein tyrosine phosphatase critically involved in the regulation of initial effector functions in B- and T-cells. The protein comprises two phosphatase (PTP) domains in its cytoplasmic region. However, whether each PTP domain has enzyme activity by itself or whether both domains are required to build up a functional enzyme is unclear. We have studied different constructions of human CD45 comprising the two PTP domains, both separately and as a single protein, fused to maltose-binding protein (MBP). In apparent contrast with previous studies, we show that the first PTP domain of CD45 (when fused to MBP) may be a viable phosphatase in the absence of the second domain. Phosphatase activity resides in the monomeric form of the protein and is lost after proteolytic cleavage of the fusion partner, indicating that MBP specifically activates the first PTP domain. Furthermore, changes in the optimal pH for activity with respect to wild-type CD45 suggest that protein-protein interactions involving residues in the neighbourhood of the catalytic site mediate enzyme activation
Frontal polymerization of diurethane diacrylates
This work deals with the preparation of poly(urethane acrylates) by using two different polymerization techniques. Namely, the classical batch procedure has been compared with frontal polymerization (FP). A thorough study on the effect of initiator type, concentration, and on the velocity of the front and its maximum temperature has been carried out. Moreover, two different synthetic ways have been studied: the one step poly(urethane acrylate) preparation starting directly from 1,6 diisocyanato hexane and 2-hydroxyethyl acrylate, and the two step procedure consisting of the synthesis of the corresponding diurethane diacrylate and of its subsequent polymerization. The first method has the advantage of being faster but some caution is necessary due to the excessive heat that is generated if the reaction conditions are not properly chosen. The second approach requires a further step but has the advantage of being more controlled. DSC analysis did not show any significant difference by comparing the thermal properties of the materials obtained by the two techniques (batch and FP). However, since FP runs are very easy and fast to be performed, FP should be seriously taken into proper account when these materials have to be prepared
Frontal polymerization of diurethane diacrylates
This work deals with the preparation of poly(urethane acrylates) by using two different polymerization techniques. Namely, the classical batch procedure has been compared with frontal polymerization (FP). A thorough study on the effect of initiator type, concentration, and on the velocity of the front and its maximum temperature has been carried out. Moreover, two different synthetic ways have been studied: the one step poly(urethane acrylate) preparation starting directly from 1,6 diisocyanato hexane and 2-hydroxyethyl acrylate, and the two step procedure consisting of the synthesis of the corresponding diurethane diacrylate and of its subsequent polymerization. The first method has the advantage of being faster but some caution is necessary due to the excessive heat that is generated if the reaction conditions are not properly chosen. The second approach requires a further step but has the advantage of being more controlled. DSC analysis did not show any significant difference by comparing the thermal properties of the materials obtained by the two techniques (batch and FP). However, since FP runs are very easy and fast to be performed, FP should be seriously taken into proper account when these materials have to be prepare
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