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Structural and Functional Characterization of Enzymes in COG3964 of the Amidohydrolase Superfamily: From Sequence to Structure to FunctionOrnelas, Argentina 1982- 14 March 2013 (has links)
The Amidohydrolase Superfamily (AHS) of enzymes is one of the most structurally and functionally studied groups of biological catalysts, exquisitely designed to carry out an extensive number of reactions defined by a similar reaction mechanism. There are approximately 11,000 genes coding for AHS proteins from about 2,100 sequenced organisms. Sequence information for these genes has been catalogued in databases, the most instrumental being the National Center for Biotechnology Information (NCBI). Despite the accessible information organized in genomic databases, there is still an extensive problem of reliability in the functional annotation of gene products assigned to the AHS.
Proteins in COG3964 of the AHS have been functionally identified as dihydroorotases and adenine deaminases. Eight proteins within three group families of COG3964 have been purified and fail to demonstrate the functionally annotated activity. A library of compounds developed by functional-group modifications was compiled and tested with these enzymes. A group of enzymes within COG3964 demonstrates the ability to hydrolyze stereospecific acetylated alpha-hydroxyl carboxylates. Substrate profiles were constructed for enzymes belonging to group 6 of COG3964. Atu3266, Oant2987 and RHE_PE00295 hydrolyze the R-isomers of a library of alpha-acetyl carboxylates of which acetyl-R-mandelate is the best substrate with catalytic efficiencies of 10^5 M^-1s^-1. This compound was identified after a series of modifications from a low-activity compound (V/K = 4 M^-1s^-1). Methylphosphonate analogs of acetyl-R-mandelate and N-acetyl-D-phenyl glycine are inhibitors of enzymes in group 6. The structure of Atu3266 was used in docking experiments to assess the selectivity of R- enantiomers over their S- counterparts. An additional group of orthologues share less than 40% sequence similarity to enzymes from group 6. EF0837, STM4445 and BCE_5003 from group 2 show significantly lower rates for the hydrolysis of alpha-acetyl carboxylates, including acetyl-R-mandelate, hydrolyzed at values of kcat/Km = 10^3 M^-1s^-1. This is also the only active compound for EF0837. Xaut_0650 and blr3349 from group 7 of COG3964 demonstrate less than 30% identity to enzymes in groups 2 and 6. These enzymes fail to hydrolyze any compound from an extended library of compounds.
An annotated selenocysteine synthase gene (SelA) from COG1921 has been identified as a gene neighbor to almost every amidohydrolase from COG3964. Atu3263, Oant2990 and EF0838 are pyridoxal-5’-phosphate dependent enzymes that were purified and assayed with D- and L- amino acids. Initial thermal-shift fluorescence assays determined that in the presence of D-cysteine, the proteins were denatured at lower temperatures.
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The Role of Cytoskeletal Morphology in the Nanoorganization of SynapseKaliyamoorthy, Venkatapathy January 2016 (has links) (PDF)
Synapse is the fundamental unit of synaptic transmission. Learning, memory and neurodegenerative diseases of the brain are attributed to the maintenance and alteration in synaptic connections. The efficiency for synaptic transmission depends on how well the post synapse receives the signals from the presynapse; this in turn depends on the receptors present in the post synaptic density (PSD). PSD is present in the post synapse right opposite to the neurotransmitter release site in presynapse (active zone) is an indispensable part of the synapse. The PSD is comprised of receptors and scaffold proteins, which is ultimately supported by the actin cytoskeleton of the dendritic spines. Cytoskeletal dynamics is shown to influence the structural plasticity of spine and also PSD, but how it regulates the dynamicity of the synaptic transmission is not completely understood. Here we studied the influence of actin depolymerisation on sub synaptic organization of an excitatory synapse. In order to study the organization of the synapse at molecular resolution, the conventional microscopy cannot be employed due to the limit of diffraction.
Super resolution microscopy circumvents this diffraction limitation. In this study we have used custom built fluorescence microscope with Total Internal Reflection Fluorescence (TIRF) modality to observe the nanometre sized structures inside spines of mouse hippocampal primary neurons. The setup was integrated with Metamorph imaging software for both operating the microscope and imaging acquisition purpose with a separate appropriate laser system. This setup was successful in achieving the lateral resolution of ~30nm and axial resolution of ~51nm. Over all we were able to observe the loss of spines and significant reduction in area of nanometer sized protein clusters in postsynaptic density with in the spines of latrunculin A treated mouse hippocampal primary neurons compared to the native neurons. Along with the morphological alterations in neurons we also observed the changes in nanoscale organization of few key molecules in the postsynaptic density.
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