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Wiseman B.,University of Manitoba | Carpena X.,Barcelona Institute for Research in Biomedicine | Carpena X.,Institute Of Biologia Molecular | Feliz M.,University of Barcelona | And 6 more authors.
Journal of Biological Chemistry | Year: 2010

Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis involves its conversion to isonicotinyl-NAD, a reaction that requires the catalase-peroxidase KatG. This report shows that the reaction proceeds in the absence of KatG at a slow rate in a mixture of INH, NAD+, Mn2+, and O2, and that the inclusion of KatG increases the rate by >7 times. Superoxide, generated by either Mn 2+- or KatG-catalyzed reduction of O2, is an essential intermediate in the reaction. Elimination of the peroxidatic process by mutation slows the rate of reaction by 60% revealing that the peroxidatic process enhances, but is not essential for isonicotinyl-NAD formation. The isonicotinyl-NAD.+ radical is identified as a reaction intermediate, and its reduction by superoxide is proposed. Binding sites for INH and its co-substrate, NAD+, are identified for the first time in crystal complexes of Burkholderia pseudomallei catalase-peroxidase with INH and NAD + grown by co-crystallization. The best defined INH binding sites were identified, one in each subunit, on the opposite side of the protein from the entrance to the heme cavity in a funnel-shaped channel. The NAD+ binding site is ∼20 Å from the entrance to the heme cavity and involves interactions primarily with the AMP portion of the molecule in agreement with the NMR saturation transfer difference results. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Grillo M.,Institute Of Biologia Molecular | Furriols M.,Institute Of Biologia Molecular | Casanova J.,Institute Of Biologia Molecular | Luschnig S.,University of Zürich
Genetics | Year: 2011

Early embryogenesis in Drosophila melanogaster is controlled by maternal gene products, which are deposited in the egg during oogenesis. It is not well understood how maternal gene expression is controlled during germline development. pipsqueak (psq) is a complex locus that encodes several nuclear protein variants containing a PSQ DNA-binding domain and a BTB/POZ domain. Psq proteins are thought to regulate germline gene expression through epigenetic silencing. While psq was originally identified as a posterior-group gene, we show here a novel role of psq in embryonic terminal patterning. We characterized a new psq loss-of-function allele, psqrum, which specifically affects signaling by the Torso (Tor) receptor tyrosine kinase (RTK). Using genetic epistasis, gene expression analyses, and rescue experiments, we demonstrate that the sole function impaired by the psqrum mutation in the terminal system is an essential requirement for controlling transcription of the tor gene in the germline. In contrast, the expression of several other maternal genes, including those encoding Tor pathway components, is not affected by the mutation. Rescue of the psqrum terminal phenotype does not require the BTB/POZ domain, suggesting that the PSQ DNA-binding domain can function independently of the BTB/POZ domain. Our finding that tor expression is subject to dedicated transcriptional regulation suggests that different maternal genes may be regulated by multiple distinct mechanisms, rather than by a general program controlling nurse-cell transcription. Copyright © 2011 by the Genetics Society of America.

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