Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The bacterial flagellum is a motility apparatus in which a long helical filament--the propeller--is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.
Mol Microbiol 1996 Jan
PMID:Flagellar assembly in Salmonella typhimurium. 882 31

AM1 quantum mechanical reaction coordinate (RC) calculations were run to simulate the rate-limiting deacylation (hydrolysis) reaction for a series of para-X-PhC(O)NHCH2-C(Y)-S-papain intermediates, where X = OCH3, CH3, H, Cl, NO2 and Y = O (thioester) or S (dithioester), for which a large body of structural, kinetic, and spectroscopic data is available. Several reaction zones, in particular the so-designated Large Zone and Small Zone, were extracted for these RC simulations from the fully solvated and energy-minimized X-ray crystal structure of papain (pdb9pap) bound to the appropriate substrate moiety. The major structural difference between these two zones was the absence of the oxyanion hole in the latter. For both the thioester and dithioester cases, the calculated Ea value associated with the parent (X = H) acyl-enzyme intermediate was lower by ca. 10 kcal/mol for the Large Zone than for the Small Zone. The magnitude of this difference suggests that the oxyanion hole plays a functional if not essential role in stabilizing the anionic tetrahedral intermediate with the cysteine proteases. The calculated Ea value was lower by ca. 10 kcal/mol for the thioester [-C(O)-S-] than for the corresponding dithioester [-C(S)-S-], in qualitative agreement with kinetic data for this series of substrates which reveal that the specific rate constant for deacylation k3 is ca. 60 times larger for the former. This difference is also consistent with both AM1 and 6-31G* calculations on model intermediates, which indicate that the weaker polarity of the dithioester compared with the thioester [i.e., -C(<--S)-S-versus-C(-->O)-S-] renders the former a much poorer site for nucleophilic attack. The anionic tetrahedral intermediate is energetically more stable for the dithioester than for the corresponding thioester, a finding that is discussed in terms of its kinetic and mechanistic implications. The mode of attack by the H2O nucleophile is "concerted" rather than "sequential" in terms of the order of proton abstraction by His-159 and nucleophilic attack on the acyl-enzyme intermediate. While the presumably key Sthiol . . . N nonbonded contact remained almost constant (ca. 2.90 A) up to formation of the [TS] structure, the substrate torsion angles phi and psi rotated significantly as the hybridization around the reaction site transforms from sp2 to sp3 during formation of the tetrahedral intermediate. The AM1-calculated frontier molecular orbitals for model thioester and dithioester acyl-enzyme intermediates generally associate the HOMOs with the reaction site and the LUMOs with the benzamide moiety. Computer graphics images corroborate our view that, in relation to the Sthiol . . . N interaction, the HOMOs and LUMOs should be identified, respectively, with Sthiol and N rather than the reverse, as suggested by other workers.
J Mol Graph 1996 Apr
PMID:Molecular modeling of substrate-enzyme reactions for the cysteine protease papain. 883 73

For most large phages of both Gram-positive and Gram-negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage-encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non-specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual-start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N-terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysiseffector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a 'hole' in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual-start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.
Mol Microbiol 1996 Aug
PMID:Two beginnings for a single purpose: the dual-start holins in the regulation of phage lysis. 887 31

Exciton level structure, homogenous absorption and hole-burning spectra were calculated for different models of bacteriochlorophyll aggregation in chlorosomal antennae of green bacteria. It was demonstrated that none of the earlier proposed models of noninteracting linear bacteriochlorophyll aggregates and linear bacteriochlorophyll chains assembling in tubular aggregates with high density of packing, exhibits the in vivo exciton level structure revealed by hole-burning experiments on intact cells of green bacteria. The models of linear exciton-coupled bacteriochlorophyll chains with a low packing density, approximating that in vivo, were proposed as alternative, to obtain the main spectral features found in natural antennae.
Biochem Mol Biol Int 1996 Oct
PMID:Structure of bacteriochlorophyll aggregates in chlorosomes of green bacteria: a spectral hole burning study. 889 46

All or part of the alpha-esterase gene cluster in Drosophila melanogaster has been isolated by screening a YAC clone that spans cytological region 84D3-10 with consensus carboxyl/cholinesterase oligonucleotides. The cluster encompasses 11 putative esterase genes within 65 kb of genomic DNA and is one of the largest clusters of related protein-coding genes yet reported in Drosophila. The cluster must include the gene encoding the major alpha-esterase isozyme, EST9, which has previously been mapped to 84D3-5. It probably also includes the genes encoding the EST23, MCE and ALI esterases that have previously been mapped to 84D3-E2. The latter three are homologs of genes involved in organophosphate insecticide resistance in the sheep blowfly, Lucilia cuprina and the housefly, Musca domestica. Sequencing of one of the putative esterase genes in the Drosophila cluster, alpha E1, shows that it would encode features characteristic of an active carboxyl/cholinesterase, including the so-called catalytic triad, the nucleophilic elbow and oxyanion hole. It also shows that the closest relative of alpha E1 amongst previously published esterase sequences is ESTB1, which confers organophosphate resistance in Culex mosquitoes. We argue that we have cloned the D. melanogaster version of a major cluster of esterase genes which have variously mutated to confer organophosphate resistance in diverse Diptera.
Insect Biochem Mol Biol 1996 Mar
PMID:Molecular cloning of an alpha-esterase gene cluster on chromosome 3r of Drosophila melanogaster. 890 May 95

The three-dimensional interaction of the enzyme-activated (suicide) inhibitor AA 231-1 [N-(2-chloromethyl)-3, 3-difluoro-azetidin-2-one] with human leukocyte elastase has been studied using computer graphics and molecular mechanics. Systematic conformational analyses and energy minimizations have been performed for the inhibitor AA 231-1 and its presumed complexes formed during the enzymatic process of inactivation, i.e., the Michaelis complex, the acyl-enzyme, and the inactivated enzyme with the covalently bound inhibitor. The beta-lactam ring characteristics of modeled AA 231-1 were in agreement with crystallographic data of related structures. Lowest energy conformations were found when the angle between the planes of the beta-lactam ring and that of its phenyl substituent was about -60 or 60 degrees. To study the interaction with the enzyme, the enzyme-inhibitor complexes were constructed by docking the inhibitor in the active site using enzyme coordinates from an X-ray crystallographic structure. The whole enzyme structure was used for conformational analyses and energy mechanics. Favorable conformations for the Michaelis complex have been obtained in which the carbonyl oxygen of the inhibitor was located in the oxyanion hole and the hydroxyl of Ser195 was in position to interact with the beta-lactam carbonyl carbon on the alpha face of AA 231-1. Simulations of the approach of the benzylic carbon by the nucleophilic amino acid His40 or His57 through an SN2 displacement on the halomethyl group of AA 231-1 were performed. The results agreed with the alkylation of the imidazole nitrogen N epsilon 2 of His57 leading to the inactivated enzyme (bis-adduct form).
J Mol Graph 1996 Jun
PMID:Interaction of human leukocyte elastase with a N-aryl azetidinone suicide substrate: Conformational analyses based on the mechanism of action of serine proteinases. 890 43

Butyrylcholinesterase [BuChE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BuChE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BuChE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BuChE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BuChE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.
Mol Pharmacol 1996 Dec
PMID:Overlapping drug interaction sites of human butyrylcholinesterase dissected by site-directed mutagenesis. 896 62

All four spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 contain a common structural element called the snRNP core. This core is assembled from the common snRNP proteins and the small nuclear RNA (snRNA). We have used electron microscopy to study the structure of two intermediates of the snRNP core assembly pathway: (1) the (E.F.G) protein complex, which contains only the smallest common proteins E, F and G; and (2) the subscore of U5 snRNP, in which the U5 RNA and the common proteins D1 and D2 are bound to the (E.F.G) protein complex. The general structure of the subscore was found to resemble that of the complete snRNP core, which contains the components of the subscore plus the common proteins B/B' and D3. Both the complete snRNP core and subscore particles are globular, with diameters of 7 to 8 nm. They show a characteristic accumulation of stain at the centre. However, some subscore images showed nicked outlines not seen with the complete snRNP cores. The (E.F.G) protein complex appeared as a ring, with an outer diameter of about 7 nm and a central hole 2 nm across. The molecular dimensions of the E, F and G proteins imply that the thickness of the (E.F.G) ring structure is only about 2 nm. Comparison of the (E.F.G) structure complex with the snRNP core and subcore structures implicates that a flat side of the ring-shaped (E.F.G) complex provides the assembly site(s) for the other components of the snRNP during core assembly: first for the D1 and D2 proteins (and probably the snRNA) during subscore formation, and then for the B/B' and D3 proteins in the completion of the snRNP core particle.
J Mol Biol 1997 Jan 17
PMID:Electron microscopy of assembly intermediates of the snRNP core: morphological similarities between the RNA-free (E.F.G) protein heteromer and the intact snRNP core. 902 Sep 71

Most examples of positive selection inferred from nucleotide sequence data involve hostpathogen interactions. However, positive selection also promotes the divergence of proteins mediating sperm-egg recognition in marine invertebrates. The abalone spermatozoon has a large acrosomal vesicle containing two proteins of 16 kDa and 18 kDa. Lysin, the 16-kDa protein, exhibits species-specificity in dissolving a hole in the egg vitelline envelope through which the sperm swims to reach the egg plasma membrane. The 18-kDa protein coats the sperm acrosomal process and probably mediates fusion of the two gametes. In this review, we compare sequences of both proteins from five species of California abalones. Both proteins show extensive divergence which has been promoted by positive Darwinian selection. The ratios of nonsynonymous to synonymous nucleotide substitutions may be the highest yet discovered for full-length sequences. Although extensive divergence has occurred, there is conservation of the shape and polarity of residues in both proteins. The two acrosomal proteins arose by a gene duplication followed by their extensive divergence. Five hypotheses are presented which attempt to explain the nature of the unknown selective force responsible for the robust positive selection. The positive selection may, in some unknown way, be related to the establishment of prezygotic barriers to reproduction. Because positive selection promotes the divergence of unrelated, species-specific gamete recognition proteins in both abalones and sea urchins, we predict that positive selection may be a general phenomenon in the evolution of gamete recognition systems in marine invertebrates.
J Mol Evol 1997
PMID:Positive Darwinian selection on two homologous fertilization proteins: what is the selective pressure driving their divergence? 907 Oct 7

Although gene delivery to the pulmonary circulation has both experimental and therapeutic potential, the delivery methods, distribution of transgene, and subsequent inflammatory response have been poorly characterized to date. To address these issues, we utilized a 0.76-mm OD (outside diameter) end hole catheter inserted into the internal jugular vein of adult Sprague-Dawley rats, directing the tip into a pulmonary capillary wedge position. We then compared infusion of polycationic lipid:DNA complexes to replication-defective adenovirus with respect to magnitude and distribution of transgene expression using either chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP) reporter genes. Both lipid:DNA and adenovirus resulted in detectable transgene expression, though maximum lung CAT activity using lipid (gamma AP-DLRIE/DOPE) was approximately 2% of maximum activity using adenovirus (Ad-CAT). Further characterization of expression after transfection with 10(8) pfu (plaque forming units) of Ad-CAT demonstrated persistence of transgene for at least 14 days (lung CAT activity 27% of maximum). Alkaline phosphatase staining demonstrated that both large and small pulmonary arteries as well as the alveolar wall expressed transgene. Although little inflammatory response was detected in conduit arteries, a predominantly mononuclear cell infiltrate surrounded small pulmonary arteries as well as the alveolar spaces in transfected areas of lung. We conclude that percutaneous catheter-mediated gene delivery to the pulmonary circulation in rats using non-viral and viral vectors is feasible. Although an inflammatory response to first generation replication-defective adenovirus was detected, it appeared to be largely restricted to the distal pulmonary circulation and airspace. This technique should prove useful for investigations requiring overexpression of novel genes in the pulmonary artery wall, and could ultimately be used to develop gene-based therapies for pulmonary vascular diseases.
Am J Respir Cell Mol Biol 1997 Jun
PMID:In vivo gene delivery to the pulmonary circulation in rats: transgene distribution and vascular inflammatory response. 919 65


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