Kinetic and oligomeric study of Leishmania braziliensis nicotinate/nicotinamide mononucleotide adenylyltransferase.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in redox reactions and oxidative stress defense system. Furthermore, the NAD is used as the substrate by a protein that regulates cell functions important for DNA repair, Other Recombinant Proteins genetic and signal transduction, among many others. NAD biosynthesis can be solved through de novo and salvage pathways, which converge at a common step is catalyzed by a nicotinate / nicotinamide mononucleotide adenylyltransferase (NMNAT EC: 2.7.7.1/18). 

Here, we report kinetic characterization of NMNAT of Leishmania braziliensis (LbNMNAT), one of the etiologic agent of leishmaniasis, a parasitic disease that is relevant. Expression and purification of recombinant proteins homogeneous 6xHis-LbNMNAT done and kinetic studies, which included analysis of Km, Vmax, kcat and equilibrium constants (KD) for both forward and reverse reactions, is completed.

Oligomeric state of the recombinant protein 6xHis-LbNMNAT studied by size exclusion chromatography. Our results show the highest and lowest Km values ​​for ATP and NAD, respectively. According to KD calculated, preferably NAD pyrophosphorolytic cleavage in vitro. In addition, the recombinant protein 6xHis-LbNMNAT shows the state of monomer, although showing structural elements involved in subunit interaction potential. 

Overall, our results show important differences from protein LbNMNAT in relation to human orthologs HsNMNAT1-3. These differences are preliminary findings that should continue to eventually propose promissory NMNAT as pharmacological targets in L. braziliensis.

Based radical photodecarboxylases photoinactivation of fatty acids.

photodecarboxylases fatty acids (FAP) is a recently discovered family of enzymes FAD-containing light-activated, which converts fatty acids to n-alkane / alkene with potential applications in the manufacturing and specialty chemicals and fuels. poor catalytic stability FAPs however major limitations.

 Here, we describe the methodology to purify samples of a stable and homogeneous catalysts Chlorella variabilis NC64 (CvFAP) from Escherichia coli. We show however that blue-exposure, whA recombinant FAPich is required for activity Parasite Recombinant Proteins photodecarboxylase, also leads to irreversible inactivation of the enzyme, especially in the absence of the substrate palmitate. Photoinactivation associated with the formation of protein based organic radical, which is observed by EPR spectroscopy.

 For photoinactivation presses, we are ready stable and catalytically active FAP in the dark