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At least 19 recordsLinked to original sources
PubMed2001

Tyrosine phosphorylation of beta-dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins.

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. However, it remains unknown whether tyrosine-phosphorylated beta-dystroglycan interacts with SH2 domain containing proteins. Here, we show that the tyrosine phosphorylation of beta-dystroglycan is constitutively elevated in v-Src transformed cells. We next reconstituted this phosphorylation event in vivo by transiently coexpressing wild-type c-Src with a fusion protein containing full-length beta-dystroglycan. Our results demonstrate that Src-induced tyrosine phosphorylation of beta-dystroglycan is strictly dependent on the presence of a PPxY motif at its extreme C-terminus. In the nonphosphorylated state, this PPxY motif is normally recognized as a ligand by the WW domain; phosphorylation at this site blocks the binding of certain WW domain containing proteins. Using a GST fusion protein carrying the cytoplasmic tail of beta-dystroglycan, we identified five SH2 domain containing proteins that interact with beta-dystroglycan in a phosphorylation-dependent manner, including c-Src, Fyn, Csk, NCK, and SHC. We localized this binding activity to the PPxY motif by employing a panel of beta-dystroglycan-derived phosphopeptides. In addition, tyrosine phosphorylation of beta-dystroglycan in vivo resulted in the coimmunoprecipitation of the same SH2 domain containing proteins, and this binding event required the beta-dystroglycan C-terminal PPxY motif. We discuss the possibility that tyrosine phosphorylation of the PPxY motif within beta-dystroglycan may act as a regulatory switch to inhibit the binding of certain WW domain containing proteins, while recruiting SH2 domain containing proteins.

3T3 Cells↗
PubMed1997

The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction.

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.

Amino Acid Sequence↗
PubMed2004

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination.

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.

Alkyl and Aryl Transferases↗
PubMed1994

Active site mapping of affinity-labeled rat oxidosqualene cyclase.

Rat liver oxidosqualene cyclase (OSC), a 78-kDa membrane-bound enzyme, was purified and labeled with the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene (Abe, I., Bai, M., Xiao, X.-Y., and Prestwich, G. D. (1992) Biochem. Biophys. Res. Commun. 187, 32-38). A 6-kDa CNBr peptide was separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane. The sequence of the first 30 amino acids of this peptide were determined by Edman degradation and showed unexpectedly high similarity to the fungal OSC from Candida albicans (50% identity with Arg413-Val442) and to the bacterial squalene cyclase from Alicyclobacillus (formerly Bacillus) acidocaldarius (37% identity with Lys356-Leu385). Further, radioanalysis clearly established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala or DDTAEA) were equally labeled by the irreversible inhibitor. This result provides the first information on the structural details of the active site of OSC and shows for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of sesquiterpene cyclases and prenyl transferases.

Affinity Labels↗
PubMed2006

Diastereoselective synthesis of novel iminosugar-containing UDP-Galf mimics: potential inhibitors of UDP-Gal mutase and UDP-Galf transferases.

Tetra-O-benzyl-D-glucofuranose was converted into uridine diphosphono-beta-Galf mimics based on an iminosugar skeleton linked to UMP by a 2-hydroxypropyl tether. The synthesis is based on the highly regio- and stereoselective cycloaddition of an original uridin-5'-yl allylphosphonate with a 1,4-dideoxy-1,4-iminogalactitol-derived cyclic nitrone, followed by the reductive elaboration of the cycloaddition product. The resulting iminogalactose-UMP conjugates are novel sugar nucleotide mimics which could be useful as inhibitors of UDP-Gal mutase and UDP-Galf transferases.

Enzyme Inhibitors↗
PubMed2006

Vitamin K deficiency reduces testosterone production in the testis through down-regulation of the Cyp11a a cholesterol side chain cleavage enzyme in rats.

Vitamin K (K) is an essential factor for the posttranslational modification of blood coagulation factors as well as proteins in the bone matrix (Gla proteins). It is known that K is not only distributed in the liver and bones but also abundantly distributed in the brain, kidney, and gonadal tissues. However, the role of K in these tissues is not well clarified. In this study, we used DNA microarray and identified the genes whose expression was affected in the testis under the K-deficient (K-def) state. The expression of genes involved in the biosynthesis of cholesterol and steroid hormones was decreased in the K-def group. The mRNA levels of Cyp11a - a rate-limiting enzyme in testosterone synthesis - positively correlated with the menaquinone-4 (MK-4) concentration in the testis. Moreover, as compared to the control (Cont) and K-supplemented (K-sup) groups, the K-def group had decreased testosterone concentrations in the plasma and testis. These results suggested that K is involved in steroid production in the testis through the regulation of Cyp11a.

Alkyl and Aryl Transferases↗
PubMed2004

The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes.

Invasive strains of Pseudomonas aeruginosa can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS. A transposon insertional mutant bank of P. aeruginosa was screened to identify P. aeruginosa genes that contribute to the ability of the bacteria to trigger host cell apoptosis. Several isolated mutants had disruptions in the fimV gene. A fimV mutant was unable to induce the expression of exoS, exoT and exsA genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion. Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface. Complementation by a fimV-containing cosmid clone restored both phenotypes to the wild-type levels. However, expression of the type III genes in the fimV mutant was not restored by the introduction of a fimV gene alone, although it restored the twitching motility. A gene downstream of fimV, encoding a tRNA pseudouridine synthase (truA) homologue, was able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. Indeed, a truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect. Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that truA controls tRNAs that are critical for the translation of type III genes or their regulators.

ADP Ribose Transferases↗
PubMed1995

Identification of the active site of vertebrate oxidosqualene cyclase.

Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane-bound enzyme, was carried out using the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently-modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp-rich motif may act as a point charge to stabilize incipient cationic charge.

Amino Acid Sequence↗
PubMed2001

Cell wall core galactofuran synthesis is essential for growth of mycobacteria.

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that glf can be knocked out in Mycobacterium smegmatis by allelic replacement only in the presence of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of replication and the M. smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the glf-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both glf and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.

Bacterial Proteins↗
PubMed1982

Loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities in 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur)-treated Escherichia coli.

Transfer RNAs from Escherichia coli B treated with either 5-fluorouracil or its analog, 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur), contain low levels of 5-fluorouracil, but are grossly deficient in pseudouridine and 5-methyluridine. The enzymes responsible for the formation of these two modified nucleosides, tRNA pseudouridine synthetase and (5-methyluridine)-methyltransferase, show substantially reduced activity levels in extracts from ftorafur- and 5-fluorouracil-treated cells relative to preparations from normal cells. When these tRNA-modifying activities are examined in vitro, both are inhibited by the addition of fluorouridine-containing tRNAs to the reaction mixtures. Pseudouridine synthetase activity shows potent inhibition. These inhibitory properties of fluorouridine-containing tRNAs, plus the inability of tRNA (5-methyluridine)-methyl-transferase to efficiently use fluorouridine-containing tRNAs as substrates, appear to account for the deficiency of 5-methyluridine and pseudouridine in tRNAs from cells containing low levels of 5-fluorouracil.

Escherichia coli↗
PubMed2001

Transcription, beta-like DNA polymerases and hypermutation.

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.

Animals↗
PubMed2000

Biosynthesis of the galactan component of the mycobacterial cell wall.

The structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-alpha-L-Rhap-(1-->3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusová, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis.

Cell Wall↗
PubMed2006

Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat.

The Shumiya cataract rat (SCR) is a hereditary cataractous strain. It is thought that the continuous occurrence of poorly differentiated epithelial cells at the bow area of the lens forms the pathophysiological basis for cataract formation in SCRs. In this study, we attempted to identify the genes associated with cataract formation in SCRs by positional cloning. Genetic linkage analysis revealed the presence of a major cataract locus on chromosome 20 as well as a locus on chromosome 15 that partially suppressed cataract onset. Hypomorphic mutations were identified in genes for lanosterol synthase (Lss) on chromosome 20 and farnesyl diphosphate farnesyl transferase 1 (Fdft1) on chromosome 15, both of which function in the cholesterol biosynthesis pathway. A null mutation for Lss was also identified. Cataract onset was associated with the specific combination of Lss and Fdft1 mutant alleles that decreased cholesterol levels in cataractous lenses to about 57% of normal. Thus, cholesterol insufficiency may underlie the deficient proliferation of lens epithelial cells in SCRs, which results in the loss of homeostatic epithelial cell control of the underlying fiber cells and eventually leads to cataractogenesis. These findings may have some relevance to other types of cataracts, inborn defects of cholesterol synthesis, and the effects of cholesterol-lowering medication.

Amino Acid Sequence↗
PubMed1993

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus.

The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M(r) of 43,622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M(r) of 34,532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10-12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.

Amino Acid Isomerases↗
PubMed2001

Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters.

Capsular polysaccharide (CPS) is a major virulence factor in Streptococcus pneumoniae. CPS gene clusters of S. pneumoniae types 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5' end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3' end of the gene cluster. Comparison of the four dTDP-L-rhamnose pathway genes (rml genes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rml genes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rml genes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5' ends and the relatively conserved rml genes at the 3' ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzx and wzy genes for all sequenced CPS gene clusters by use of motifs.

Bacterial Capsules↗
PubMed1995

Reactions of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1 aminomutase) with vinyl and acetylenic substrate analogues analysed by rapid scanning spectrophotometry.

The reactions occurring when glutamate-1-semialdehyde amino-transferase (glutamate-1-semialdehyde 2,1 aminomutase, EC 5.4.3.8) was treated with two potential mechanism-based inactivators, namely 4-aminohex-5-enoate and 4-aminohex-5-ynoate, have been investigated by monitoring rapid transient changes in the absorption spectrum of the enzyme's prosthetic group, pyridoxal 5'-phosphate. In both cases a short-lived chromophore absorbing maximally at about 500 nm was formed in a few milliseconds. In the case of the vinyl analogue (4-aminohex-5-enoate) this chromophore, considered to be a quinonoid intermediate, converted rapidly into the pyridoxamine phosphate form of the co-enzyme in a single turnover which was accompanied by negligible inactivation. However, slow inactivation of the enzyme by this compound was observed when the enzyme was made to undergo multiple turnovers by including the efficient aldehyde substrate, succinic semialdehyde. The acetylenic compound, aminohexynoate, produced more complex spectral changes with the consecutive formation of compounds absorbing maximally at 496 nm, 450 nm, 564 nm and 330 nm. The enzyme was 90% inactivated by aminohexynoate within 10 s and thereafter lost no further activity unless aldehyde substrate was added. Mechanisms and kinetic constants consistent with the observations are proposed for each compound. The observation that the acetylenic compound is a much more potent inactivator than its vinyl analogue is attributed to the occurrence of a conjugated allene as intermediate.

Acetylene↗