PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-7 (7)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
1.  Mannitol Bis-phosphate Based Inhibitors of Fructose 1,6-Bisphosphate Aldolases 
ACS Medicinal Chemistry Letters  2011;2(11):804-808.
Several 5-O-alkyl- and 5-C-alkyl-mannitol bis-phosphates were synthesized and comparatively assayed as inhibitors of fructose bis-phosphate aldolases (Fbas) from rabbit muscle (taken as surrogate model of the human enzyme) and from Trypanosoma brucei. A limited selectivity was found in several instances. Crystallographic studies confirm that the 5-O-methyl derivative binds competitively with substrate and the 5-O-methyl moiety penetrating deeper into a shallow hydrophobic pocket at the active site. This observation can lead to the preparation of selective competitive or irreversible inhibitors of the parasite Fba.
doi:10.1021/ml200129s
PMCID: PMC4018070  PMID: 24900268
Selective inhibitors; microbial enzymes; fructose bis-phosphate aldolase; glycolysis; Trypanosoma brucei
2.  Rational design, synthesis and evaluation of new selective inhibitors of microbial class II (zinc dependent) fructose bis-phosphate aldolases 
Journal of medicinal chemistry  2010;53(21):7836-7842.
We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.
doi:10.1021/jm1009814
PMCID: PMC2974033  PMID: 20929256
Fructose bisphosphate aldolase; Selective inhibitors; Hydroxamates; Candida albicans; Helicobacter pylori., Mycobacterium tuberculosis; Yersinia pestis; antibiotics
3.  A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage 
Nucleic Acids Research  2011;40(1):258-269.
All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer–tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.
doi:10.1093/nar/gkr740
PMCID: PMC3245945  PMID: 21911368
4.  Dramatic improvement of crystal quality for low-­temperature-grown rabbit muscle aldolase 
Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA–LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination.
Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA–LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination. It was concluded that the improvement of crystal quality as indicated by the higher resolution of the new RMA–LC4 complex crystals was a consequence of the introduction of new lattice contacts at lower temperature. The lattice contacts corresponded to an increased number of interactions between high-entropy side chains that mitigate the lattice strain incurred upon cryocooling and accompanying mosaic spread increases. The thermodynamically unfavorable immobilization of high-entropy side chains used in lattice formation was compensated by an entropic increase in the bulk-solvent content owing to the greater solvent content of the crystal lattice.
doi:10.1107/S1744309110011875
PMCID: PMC2864701  PMID: 20445268
rabbit muscle aldolase; improvement of crystal quality; low-complexity domain; sorting nexin 9
5.  Purification, crystallization and preliminary X-ray diffraction analysis of the Whirly domain of StWhy2 in complex with single-stranded DNA 
The Whirly domain of StWhy2 was crystallized both in the free form and in complex with single-stranded DNA.
StWhy1 and StWhy2 are members of the Whirly family of single-stranded DNA (ssDNA) binding proteins. To understand the mode of binding of the Whirly proteins to single-stranded DNA, crystals of the Whirly domains of both StWhy1 and StWhy2 in complex with single-stranded DNA were obtained by the hanging-drop vapour-diffusion method. The diffraction patterns of the StWhy1–ssDNA complex crystals displayed severe anisotropy and were of low resolution, making them unsuitable for structure determination. In contrast, the crystals of the StWhy2–ssDNA complex diffracted isotropically to 2.20 Å resolution. The crystallization and data collection to 2.20 Å resolution of StWhy2 in the free form are also reported.
doi:10.1107/S1744309108032399
PMCID: PMC2581700  PMID: 18997341
single-stranded DNA-binding proteins; protein–DNA complexes; Whirly domains; StWhy1; StWhy2; anisotropy
6.  Aldolase is essential for energy production and bridging adhesin-cytoskeletal interactions during parasite invasion of host cells 
Cell host & microbe  2009;5(4):353-364.
SUMMARY
Apicomplexan parasites are obligate intracellular pathogens that rely on actin-based motility to drive host cell invasion. Prior in vitro studies implicated aldolase in coupling actin filaments to the cytoplasmic domains of surface adhesins in the parasite. Here, we tested the essentiality of this interaction in host cell invasion. Homology modeling indicated a partial overlap of the binding surfaces between the enzyme active site and the region responsible for interaction with the microneme protein 2 cytoplasmic tail domain (MIC2t). Targeted mutagenesis delineated residues unique to each activity based on in vitro studies. Complementation of a conditional knockout (cKO) of the T. gondii aldolase gene (TgALD1) with mutants defective in either distinct function was used to test their respective roles. Our studies demonstrate aldolase is not only required for energy production, but is also essential for efficient host cell invasion based on its ability to bridge adhesin-cytoskeleton interactions in the parasite.
doi:10.1016/j.chom.2009.03.005
PMCID: PMC2683947  PMID: 19380114
aldolase; MIC2; enzyme; protein-protein interaction; motility; invasion
7.  Gene Cloning, DNA Sequencing, and Expression of Thermostable β-Mannanase from Bacillus stearothermophilus 
Applied and Environmental Microbiology  1998;64(11):4428-4432.
A DNA genomic library constructed from Bacillus stearothermophilus, a gram-positive, facultative thermophilic aerobe that secretes a thermostable β-mannanase, was screened for mannan hydrolytic activity. Recombinant β-mannanase activity was detected on the basis of the clearing of halos around Escherichia coli colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene, manF, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The manF coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in E. coli as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni2+ affinity chromatography. The values for the kinetic parameters Vmax and Km were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.
PMCID: PMC106664  PMID: 9797302

Results 1-7 (7)