Structure and catalytic regulation of Plasmodium falciparum IMP specific nucleotidase.

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.

Adaptation of Lactobacillus acidophilus to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2.

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila.

Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DATABASE: The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF. STRUCTURED DIGITAL ABSTRACT: LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].