Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.
J Mol Biol 2006 Jun 16
PMID:Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis. 1669 36

Expression of the Bacillus subtilis tryptophan biosynthetic genes trpEDCFBA and trpG, as well as a putative tryptophan transport gene (trpP), are regulated in response to tryptophan by the trp RNA-binding attenuation protein (TRAP). TRAP regulates expression of these genes by transcription attenuation and translation control mechanisms. Here we show that TRAP also regulates translation of ycbK, a gene that encodes a protein with similarities to known efflux proteins. As a likely TRAP-binding site consisting of 11 NAG repeats overlaps the ycbK translation initiation region, experiments were carried out to determine whether TRAP regulates translation of ycbK. TRAP was observed to regulate expression of a ycbK'-'lacZ translational fusion 20-fold in response to tryptophan. Binding studies indicated that TRAP binds to the ycbK transcript with high affinity and specificity. Footprint studies revealed that the central seven triplet repeats were protected by bound TRAP, while toeprint results suggest that nine triplet repeats contribute to TRAP binding. Additional toeprint and in vitro translation analyses demonstrated that bound TRAP regulates YcbK synthesis by blocking ribosome binding. We also identified two dipeptide coding minigenes between the Shine-Dalgarno sequence and start codon of ycbK. Expression of one of the minigenes modestly interfered with translation of ycbK.
Mol Microbiol 2006 Sep
PMID:The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis regulates translation initiation of ycbK, a gene encoding a putative efflux protein, by blocking ribosome binding. 1687 15

N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) catalyzes the reversible epimerization between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc). We report here the 2.0 A resolution crystal structure of the GlcNAc 2-epimerase from Anabaena sp. CH1. The structure demonstrates an (alpha/alpha)(6) barrel fold, which shows structural homology with porcine GlcNAc 2-epimerase as well as a number of glycoside hydrolase enzymes and other sugar-metabolizing enzymes. One side of the barrel structure consists of short loops involved in dimer interactions. The other side of the barrel structure is comprised of long loops containing six short beta-sheets, which enclose a putative central active-site pocket. Site-directed mutagenesis of conserved residues near the N-terminal region of the inner alpha helices shows that R57, H239, E308, and H372 are strictly required for activity. E242 and R375 are also essential in catalysis. Based on the structure and kinetic analysis, H239 and H372 may serve as the key active site acid/base catalysts. These results suggest that the (alpha/alpha)(6) barrel represents a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner alpha helices, thus forming a fine-tuned catalytic site in GlcNAc 2-epimerase.
J Mol Biol 2007 Mar 30
PMID:The central cavity from the (alpha/alpha)6 barrel structure of Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase contains two key histidine residues for reversible conversion. 1729 97

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.
J Mol Biol 2007 Sep 21
PMID:The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans. 1768 43

Hyaluronan (HA) is a large but simple glycosaminoglycan composed of repeating D-glucuronic acid, beta1-3 linked to N-acetyl-D-glucosamine beta1-4, found in body fluids and tissues, in both intra- and extracellular compartments. Despite its structural simplicity, HA has diverse functions in skeletal biology. In development, HA-rich matrices facilitate migration and condensation of mesenchymal cells, and HA participates in joint cavity formation and longitudinal bone growth. In adult cartilage, HA binding to aggrecan immobilises aggrecan, retaining it at the high concentrations required for compressive resilience. HA also appears to regulate bone remodelling by controlling osteoclast, osteoblast and osteocyte behaviour. The functions of HA depend on its intrinsic properties, which in turn rely on the degree of polymerisation by HA synthases, depolymerisation by hyaluronidases, and interactions with HA-binding proteins. HA synthesis and degradation are closely regulated in skeletal tissues and aberrant synthetic or degradative activity causes disease. The role and regulation of HA synthesis and degradation in cartilage, bone and skeletal development is discussed.
Cell Mol Life Sci 2008 Feb
PMID:Hyaluronan synthesis and degradation in cartilage and bone. 1796 30

The association between hypercholesterolemia and kidney damage has been well known for last few decades. The oxidative stress and inflammatory responses are involved in renal injury, which is upregulated in hypercholesterolemic condition. The present study is aimed to evaluate the possible effect of lupeol and its ester derivative, lupeol linoleate in renal damage associated with hypercholesterolemic rats. Hypercholesterolemia was induced in male Wistar rats by feeding them with a high cholesterol diet (HCD) comprising normal rat chow supplemented with 4% cholesterol and 1% cholic acid for 30 days. Lupeol and lupeol linoleate were supplemented (50 mg/kg body wt/day) to HCD fed rats during the last 15 days. Increased levels of renal total cholesterol, triglycerides and phospholipids, along with altered serum biochemical parameters of tissue injury indices and elevated activities of renal marker enzymes (lactate dehydrogenase and alkaline phosphatase) were noted in HCD fed rats. Elevated lipid peroxidation levels coupled with decreased antioxidant status (enzymatic and non enzymatic antioxidants) were observed in hypercholesterolemic rats, which indicate the onset of oxidative changes in the renal tissue. Renal lysosomal acid hydrolase activities (ACP, beta-Glu, beta-Gal, NAG and Cat-D) and acute phase proteins like C-Reactive protein and fibrinogen were significantly increased in HCD fed rats, which further indicates the heightening of inflammation. In addition, histopathological findings also confirmed the renal damage in hypercholesterolemic condition. Lupeol and lupeol linoleate effectively reverted the above abnormalities and was comparable with that of the control. These observations highlight the protective effect of lupeol and its ester derivative in ameliorating the renal injury associated with hypercholesterolemia.
Mol Cell Biochem 2008 Oct
PMID:Protective effect of lupeol and lupeol linoleate in hypercholesterolemia associated renal damage. 1856 36

The three-dimensional (3D) model of the human acidic mammalian chitinase (hAMCase) was constructed based on the crystal structure of the human chitotriosidase (EC 3.2.1.44, PDB code 1HKK) by using InsightII/Homology module. With the aid of molecular mechanics and molecular dynamics methods, the last refined model was obtained and further assessed by Profile-3D and Procheck, which confirms that the refined model is reliable. Furthermore, the docking results of the ligands (allosamidin and NAG(2)) into the active site of hAMCase indicate that allosamidin is a more preferred ligand than NAG(2), and that Glu119 forms hydrogen bond with allosamidin, which is in good agreement with the experimental results. From the docking studies, we also suggest that Trp10, Glu49, Asp192, and Glu276 in hAMCase are four important determinant residues in binding as they have strong van-der-Waals and electrostatic interactions with the ligand, respectively.
J Mol Model 2009 May
PMID:Analysis of a three-dimensional structure of human acidic mammalian chitinase obtained by homology modeling and ligand binding studies. 1908 22

The effects of osthol, a plant coumarin, on morphology, sugar uptake and cell wall components of Fusarium graminearum were examined in vitro by electron microscopy,(14)C-labelling and enzyme activity detection. The results revealed that osthol could inhibit the hypha growth of F. graminearum by decreasing hyphal absorption to reducing sugar. After treatment with 100 microg.mL(-1) osthol for 24 h, many hyphal fragments of F. graminearum appeared. Microscopy observation showed that the cell walls of hyphal fragments blurred and the organelles of the cells degraded with the increasing vacuoles. The N-acetyl-D-glucosamine contents and chitinase activity both increased when hypha were treated with 100 microg.mL(-1) osthol, whereas the activity of beta-1,6-glucanase remained unchanged. When F. graminearum fed with (14)C glucose was treated with 100 microg.mL(-1)osthol, glucose contents decreased to the lowest level, while the contents in non-osthol treated controls remained unchanged. These results suggested that chitinase activity might be related to glucose starvation under osthol treatment, and that the appearance of hyphae fragments maybe the results of the promoted chitinase activity which itself triggered chitin degradation.
Int J Mol Sci 2008 Mar
PMID:Fusarium graminearum growth inhibition due to glucose starvation caused by osthol. 1932 55

Crystal structures of Atlantic cod lysozyme have been solved with and without ligand bound in the active site to 1.7 and 1.9 A resolution, respectively. The structures reveal the presence of NAG in the substrate binding sites at both sides of the catalytic Glu73, hence allowing the first crystallographic description of the goose-type (g-type) lysozyme E-G binding sites. In addition, two aspartic acid residues suggested to participate in catalysis (Asp101 and Asp90) were mutated to alanine. Muramidase activity data for two single mutants and one double mutant demonstrates that both residues are involved in catalysis, but Asp101 is the more critical of the two. The structures and activity data suggest that a water molecule is the nucleophile completing the catalytic reaction, and the roles of the aspartic acids are to ensure proper positioning of the catalytic water.
Cell Mol Life Sci 2009 Aug
PMID:Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism. 1954 50

The binding abilities of human C-reactive protein (CRP) with the C-terminal fibrinogen-like (FBG) domain and the full-length form of human M-ficolin were investigated by pull-down and zonal affinity chromatography analyses. Pull-down assays using an N-acetyl-D-glucosamine (GlcNAc)-agarose column demonstrated that CRP binds to the trimeric FBG domains, and that the GlcNAc-binding ability of the FBG domain is unaffected by CRP binding. Interestingly, the full-length M-ficolin, comprising the N-terminal collagen-like (COL) and C-terminal FBG domains, displayed lower affinity for CRP, and the monomeric FBG domain showed virtually no binding to CRP, as qualitatively judged by zonal affinity chromatography using a GlcNAc column. These results indicated that CRP binding requires the trimeric form of the FBG domain, and that the presence of the COL domain reduces the interaction between CRP and M-ficolin. In addition, pull-down assays using a histidine-tag affinity column demonstrated that neither the full-length M-ficolin nor the trimeric FBG domains, immobilized through their C-terminal histidine tags, showed any affinity for CRP, indicating that the CRP binding site is located near Ala326 at the C-terminus of M-ficolin, spatially close to a neck region (around Pro115) between the FBG and COL domains. From these findings, we concluded that CRP binding is enhanced by conformational bending at the neck region of M-ficolin, to avoid steric hindrance by the COL domain. Such a situation may be generated by oligomeric M-ficolin binding to surfaces with widely distributed ligands, such as pathogens.
Mol Immunol 2009 Dec
PMID:Binding site of C-reactive protein on M-ficolin. 1985 18


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