bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical analysis of a thermostable tryptophan synthase from a hyperthermophilic archaeon
Authors: Tang Xiao‐Feng , Ezaki Satoshi , Atomi Haruyuki , Imanaka Tadayuki
Published: March 2003

Pyridoxal 5′‐phosphate‐dependent tryptophan synthase catalyzes the last two reactions of tryptophan biosynthesis, and is comprised of two distinct subunits, α and β. TktrpA and TktrpB, which encode the α subunit and β subunit of tryptophan synthase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, were independently expressed in Escherichia coli and their protein products were purified. Tryptophan synthase complex (Tk‐TS complex), obtained by heat treatment of a mixture of the cell‐free extracts containing each subunit, was also purified. Gel‐filtration chromatography revealed that Tk‐TrpA was a monomer (α), Tk‐TrpB was a dimer (β2), and Tk‐TS complex was a tetramer (α2β2). The Tk‐TS complex catalyzed the overall αβ reaction with a specific activity of 110 µmol Trp per µmol active site per min under its optimal conditions (80 °C, pH 8.5). Individual activity of the α and β reactions of the Tk‐TS complex were 8.5 µmol indole per µmol active site per min (70 °C, pH 7.0) and 119 µmol Trp per µmol active site per min (90 °C, pH 7.0), respectively. The low activity of the α reaction of the Tk‐TS complex indicated that turnover of the β reaction, namely the consumption of indole, was necessary for efficient progression of the α reaction. The α and β reaction activities of independently purified Tk‐TrpA and Tk‐TrpB were 10‐fold lower than the respective activities detected from the Tk‐TS complex, indicating that during heat treatment, each subunit was necessary for the other to obtain a proper conformation for high enzyme activity. Tk‐TrpA showed only trace activities at all temperatures examined (40–85 °C). Tk‐TrpB also displayed low levels of activity at temperatures below 70 °C. However, Tk‐TrpB activity increased at temperatures above 70 °C, and eventually at 100 °C, reached an equivalent level of activity with the β reaction activity of Tk‐TS complex. Taking into account the results of circular dichroism analyses of the three enzymes, a model is proposed which explains the relationship between structure and activity of the α and β subunits with changes in temperature. This is the first report of an archaeal tryptophan synthase, and the first biochemical analysis of a thermostable tryptophan synthase at high temperature.
A novel mammalian Smt3‐specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase
Authors: Nishida Tamotsu , Tanaka Hirofumi , Yasuda Hideyo
Published: March 2003

A novel Smt3‐specific isopeptidase, SMT3IP1, was cloned using a yeast two‐hybrid screen with Smt3b as bait. The clone, named SMT3IP1 (Smt3‐specific isopeptidase 1), which bound to Smt3b but not SUMO‐1 in the two‐hybrid system, was distantly related to budding yeast Saccharomyces cerevisiae Ulp1, human SENP1 or human SUSP1. The catalytic domains in the C‐terminal region were very similar, but the N‐terminal region was quite different to other enzymes. The cysteine, histidine and asparatic acid residues in the catalytic domains were conserved. SMT3IP1 expressed by the baculovirus‐expression system had the ability to cleave SUMO‐1 or Smt3b from SUMO‐1/RanGAP1 or Smt3b/RanGAP1 conjugates, respectively, and the activity was a little stronger towards the Smt3b conjugate than towards the SUMO‐1 conjugate. Furthermore, the enzyme bound more strongly to Smt3a and Smt3b than to SUMO‐1 in vitro. The enzyme did not cleave Nedd8 from Nedd8/cullin‐1. Nor did it cleave ubiquitin from ubiquitinated p53. SMT3IP1 was localized almost exclusively at the nucleolus during interphase. The N‐terminal sequence was responsible for the nucleolar localization of this enzyme. Whether SMT3IP1 functions in the nucleolus or just stays there before it functions in the nucleus, as shown in the case of CDC14 phosphatase, remains to be elucidated.
Domains of Brn‐2 that mediate homodimerization and interaction with general and melanocytic transcription factors
Authors: Smit Darren J. , Smith Aaron G. , Parsons Peter G. , Muscat George E. O. , Sturm Richard A
Published: March 2003

The class III POU gene brn‐2, encoding the Brn‐2/N‐Oct‐3 transcription factor, is widely expressed in the developing mammalian central nervous system. Brn‐2 has also been found to regulate the melanocytic phenotype with N‐Oct‐3 DNA binding activity elevated in malignant melanoma, however, its mode of action is yet to be defined. The functional role of the Brn‐2 transcription factor has been investigated through the analysis of protein–protein interactions it forms with a number of basal and melanocytic transcriptional regulatory proteins. In vivo interactions were tested by gene‐cotransfection using the mammalian GAL4–Herpes Simplex viral protein 16 (VP16) two hybrid formation and direct protein binding by in vitro glutathione S‐transferase (GST)‐pull down assay. The Brn‐2 protein was found to homodimerize in vivo with high affinity, using Brn‐2 deletion constructs dimer complex formation was found to be dependent on the presence of both the homeodomain and linker regions of the POU‐domain. However, the POU‐homoedomain was dispensable for the formation of the dimerization interface in one of the partner molecules but not both, when the POU‐linker region was removed the ability to interact was lost irrespective of the presence of the homeodomain. Dimerization of Brn‐2/N‐Oct‐3 was also found to occur in DNA binding assays using melanoma cell line nuclear extracts and a recently reported dimer target sequence probe, which may have significant consequences for gene regulation in melanocytic tumours. Low affinity Brn‐2 protein contacts have also been found with the basal transcription complex, including TATA binding protein (TBP) and the transcriptional coactivator p300, and with the Sox‐10 and Pax‐3 transcription factors that are known to play an important role in melanocyte cell formation.
Peroxovanadate induces tyrosine phosphorylation of phosphoinositide‐dependent protein kinase‐1
Authors: Grillo Sophie , Grémeaux Thierry , Casamayor Antonio , Alessi Dario R , Le Marchand‐Brustel Yannick , Tanti Jean‐François
Published: March 2003

Phosphoinositide‐dependent protein kinase‐1 (PDK1) is a recently identified kinase that phosphorylates and activates protein kinase B (PKB). Activation of PKB by insulin is linked to its translocation from the cytosol to the plasma membrane. However, no data are available yet concerning the localization of PDK1 in insulin‐sensitive tissue. Using isolated adipocytes, we studied the effect of insulin and of an insulin‐mimicking agent peroxovanadate on the subcellular localization of PDK1. In unstimulated adipocytes, overexpressed PDK1 was mostly cytosolic with a low amount associated to membranes. Peroxovanadate stimulation induced the redistribution of PDK1 to the membranes while insulin was without effect. This peroxovanadate effect was dependent on phosphatidylinositol 3,4,5 triphosphate [PtdIns(3,4,5)P3] production as inhibition of PtdIns 3‐kinase by wortmannin or deletion of the PH domain of PDK1 prevented the peroxovanadate‐induced translocation of PDK1. Further, peroxovanadate‐treatment induced a tyrosine phosphorylation of PDK1 which was wortmannin insensitive and did not require the PH domain of PDK1. An inhibitor of Src kinase (PP2) decreased the peroxovanadate‐induced PDK1 tyrosine phosphorylation and overexpression of v‐Src stimulated this phosphorylation. Mutation of tyrosine 373 of PDK1 abolished the v‐Src induced PDK1 tyrosine phosphorylation and partially reduced the effect of peroxovanadate. Our findings suggest that PDK1 could be a substrate for tyrosine kinases and identify Src kinase as one of the tyrosine kinases able to phosphorylate PDK1.
Characterization of the general odorant‐binding protein 2 in the molecular coding of odorants in Mamestra brassicae
Authors: Jacquin‐Joly Emmanuelle , Bohbot Jonathan , Francois Marie‐Christine , Cain Anne‐Hélène , Nagnan‐Le Meillour Patricia
Published: March 2003

The general odorant‐binding protein 2 of Mamestra brassicae males has been purified from antennal extracts and examined in binding assays with pheromone components of this species and a behavioral antagonist, cis‐11‐hexadecenol. The protein showed high affinity for the latter compound and no affinity for the pheromone components. In addition, expression of the protein, studied by in situ hybridization, was restricted to the long sensilla trichodea, which house the neuron that responds to cis‐11‐hexadecenol. The expression in a functionally defined population of sensilla, together with binding specificity and previous electrophysiological data, suggest an unsuspected role for the general odorant‐binding protein 2 in M. brassicae. It may be involved in the transduction process for the behavioral antagonist to which neurons are specifically tuned and always cocompartmentalized in long trichodeal hairs, with neurons responding to the major pheromonal compound, cis‐11‐hexadecenyl acetate. These data are consistent with the involvement of odorant‐binding proteins in the fine discrimination between pheromone and antagonist, which is related to avoidance of interspecific mating mistakes.
Role of arginine 177 in the MnII binding site of manganese peroxidase
Authors: Gelpke Maarten D. Sollewijn , Youngs Heather L. , Gold Michael H.
Published: March 2003

Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild‐type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady‐state and transient‐state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt‐bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second‐order rate constants for compound I reduction (k2app), and decreased first‐order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron‐transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.
Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifolii
Authors: Lee Hwan Young , An Jae Hyung , Kim Yu Sam
Published: March 2003

A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl‐CoA decarboxylase, malonyl‐CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl‐CoA. The deduced amino‐acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N‐terminal region encoding a helix‐turn‐helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered −46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR‐mediated repression effect on the expression of Pmat‐luc+ reporter fusion.
Mutational analysis of the active site of human insulin‐regulated aminopeptidase
Authors: Laustsen Palle G. , Vang Søren , Kristensen Torsten
Published: March 2003

Insulin‐regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn2+‐coordination sequence element, HEXXH‐(18–64X)‐E. A second conserved sequence element, the GXMEN motif, positioned 22–32 amino acids N‐terminal to the Zn2+‐coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH‐(18X)‐E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full‐length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild‐type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino‐acid substitutions in the GAMEN motif (G428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn2+‐binding motif (H464Y, E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased or abolished aminopeptidase activity towards the leucine‐para‐nitroanilide substrate. The results show that conservation of residues within the GAMEN and Zn2+‐binding motifs is important for IRAP enzyme activity.
Engineering the active site of ascorbate peroxidase
Authors: Çelik Ayhan , Cullis Paul M. , Sutcliffe Michael J. , Sangar Rajesh , Raven Emma Lloyd
Published: March 2003

The oxidation of a number of thioethers, namely methyl phenyl sulphide (1), ethyl phenyl sulphide (2), isopropyl phenyl sulphide (3), n‐propyl phenyl sulphide (4), p‐chlorophenyl methyl sulphide (5), p‐nitrophenyl methyl sulphide (6) and methyl naphthalene sulphide (7), by recombinant pea cytosolic ascorbate peroxidase (rAPX) and a site‐directed variant of rAPX in which the distal tryptophan 41 residue has been replaced with an alanine (W41A) has been examined. The electronic spectrum (pH 7.0, µ = 0.10 m, 25.0 °C) for the ferric derivative of W41A (λmax/nm = 411, 534, 560, 632) is indicative of an increased quantity of 6‐coordinate, high‐spin and/or 6‐coordinate, low‐spin haem compared to rAPX. Steady state oxidation of sulphides 1–4 and 7, gave values for kcat that are approximately 10‐fold and 100‐fold, respectively, higher for W41A than for rAPX. For rAPX, essentially racemic mixtures of R‐ and S‐sulphoxides were obtained for all sulphides. With the exception of sulphide 7, the W41A variant shows substantial enhancements in enantioselectivity, with R : S ratios varying between R : S = 63 : 37 (sulphides 1 and 4) and R : S = 85 : 15 (sulphide 6). Incubation of sulphide 2 with rAPX or W41A and [18O] H2O2 shows 95% (rAPX) and 96% (W41A) transfer of labelled oxygen to the substrate. Structure‐based modelling techniques have provided a fully quantitative rationalization of all the experimentally determined R : S ratios and have indicated that reorientation of the sidechain of Arg38, such that access to the haem is much less restricted, is influential in controlling the stereoselectivity for both rAPX and W41A.
Binding of p53 and its core domain to supercoiled DNA
Authors: Palecek Emil , Brázdová Marie , Brázda Václav , Palecek Jan , Billová Sabina , Subramaniam Vinod , Jovin Thomas M.
Published: March 2003

We have compared the binding of human full‐length p53 protein (p53; expressed in bacteria and insects) and its isolated core domain (p53CD, amino acids 94–312; expressed in bacteria) to negatively supercoiled (sc) DNA using gel electrophoresis and immunoblotting. Significant differences were observed; p53CD produced a relatively small and continuous retardation of scDNA, in contrast to the ladder of distinct bands formed by p53 in agarose gels. The ladder produced by full‐length protein expressed in bacteria (p53b) was similar to that observed earlier with protein expressed in insect cells (p53i). Competition between scDNAs and their linearized (lin) forms showed a preference for scDNAs by both p53 and p53CD, but the ratios characterizing the distribution of the protein between sc and lin pBluescript DNAs were substantially higher for p53 (sc/lin > 60 in p53b) than for p53CD (sc/lin ≈ 4). Strong binding of p53 to scDNA lacking the p53 consensus sequence may represent a new p53‐binding mode, which we tentatively denote supercoil‐selective (SCS) binding. This binding requires both the C‐terminal domain and the core domain. Targets of this binding may include: (a) DNA segments defined both by the nucleotide sequence and local topology, and/or (b) strand crossings and/or bending. The binding preference of p53CD for scDNA may be due to the known nonspecific binding to internal single‐stranded regions in scDNA (absent in relaxed DNA molecules) and/or to SCS binding albeit with reduced affinity due to the absence of contributions from other p53 domains.
Characterization of the reversible nature of the reaction catalyzed by sphingolipid ceramide N‐deacylase
Authors: Kita Katsuhiro , Kurita Toyohisa , Ito Makoto
Published: March 2003

Sphingolipid ceramide N‐deacylase catalyzes a reversible reaction in which the amide linkages of the ceramides of various sphingolipids are cleaved or synthesized. Hydrolysis of sphingolipids by the enzyme proceeded efficiently at acidic pH in the presence of high concentrations of detergents, whereas the reverse reaction tended to be favored at neutral pH with a decrease in the detergent concentration. Although the catalytic efficiency (Vmax/Km) of the hydrolysis and reverse reactions was changed mainly by the concentration of detergents in the reaction mixture, Vmax and Km for the reverse reaction were relatively higher than those for the forward reaction, irrespective of the detergent concentration. The reverse reaction proceeded most efficiently when the molar ratio of lyso‐sphingolipids and fatty acids was fixed at 1 : 1–2, the yield of the reaction exceeding 70–80%. The reverse and exchange (transacylation) reactions did not require ATP, CoA, metal ions or addition of organic solvents. Studies using inhibitors and chemical modifiers of the enzyme protein suggested that both the hydrolysis and condensation reactions are catalyzed at the same catalytic domain. These results indicate that the reverse hydrolysis reaction of the enzyme is unique, being completely different from those of lipases, proteases and glycosidases reported to date.
Glycoinositolphosphosphingolipids (basidiolipids) of higher mushrooms
Authors: Jennemann Richard , Geyer Rudolf , Sandhoff Roger , Gschwind Ruth M. , Levery Steven B. , Gröne Hermann‐Josef , Wiegandt Herbert
Published: March 2003

The basidiolipids of six mushroom species, i.e. the basidiomycetes Amanita virosa (engl., death cup), Calvatia exipuliformis (engl., puffball), Cantharellus cibarius (engl., chanterelle), Leccinum scabrum (engl., red birch boletus), Lentinus edodes (jap., Shiitake), and Pleurotus ostreatus (engl., oystermushroom), were isolated, and their chemical structures investigated. All glycolipids are structurally related to those of the Agaricales (engl., field mushroom). They are glycoinositolphosphosphingolipids, their ceramide moiety consisting of t18:0‐trihydroxysphinganine and an α‐hydroxy long‐chain fatty acid. In contrast to a previous study [Jennemann, R., Bauer, B.L., Bertalanffy, H., Geyer, R., Gschwind, R.M., Selmer, T. & Wiegandt, H. (1999) Eur. J. Biochem.259, 331–338], the glycoside anomery of the hexose (mannose) connected to the inositol of all investigated basidiomycete glycolipids, including the basidiolipids of Agaricus bisporus, was determined unequivocally to be alpha. Therefore, the root structure of all basidiolipids consists of α‐dManp‐2Ins1‐[PO4]‐Cer. In addition, for some mushroom species, the occurrence of an inositol substitution position variant, α‐Manp‐4Ins1‐[PO4]‐Cer, is shown. The carbohydrate of chanterelle basidiolipids consists solely of mannose, i.e. Cc1, Manα‐3 or ‐6Manα; Cc2, Manα‐3(Manα‐6)Manα‐. All other species investigated show extension of the α‐mannoside in the 6‐position by β‐galactoside, which, in some instances, is α‐fucosylated in 2‐position (Fucα‐2)Galβ‐6Manα‐. Further sugar chain elongation at the β‐galactoside may be in 3‐ and/or 6‐position by α‐galactoside, e.g. Ce4, Po2, Galα‐3‐(Galα‐6)(Fucα‐2)Galβ‐6Manα‐, whereas A. virosa, Av‐3, has a more complex, highly α‐fucosylated terminus, Galα‐3 (Fucα‐2)(Fucα‐6)Galα‐2(Galα‐3)Galβ‐6Manα‐. L. edodes basidiolipids show further elongation by α‐mannoside, e.g. Le3, Manα‐2Manα‐6Galα‐3(Fucα‐2)Galβ‐6Manα‐, C. exipuliformis glycolipid by α‐glucoside, i.e. Ce3, Glcα‐6Galβ‐6Manα‐. Basidiolipid Ls1 from L. scabrum, notably, has a 3‐α‐mannosylated α‐fucose, i.e. Galα‐6(Manα‐3Fucα‐2)Galα‐6Galβ‐6Manα‐. In conclusion, basidiolipids, though identical in their ceramide constitution, display wide and systematic mushroom species dependent variabilities of their chemical structures.
A comparative study of the asparagine‐linked oligosaccharides on siglec‐5, siglec‐7 and siglec‐8, expressed in a CHO cell line, and their contribution to ligand recognition
Authors: Freeman Sylvie , Birrell Helen C. , D'Alessio Karla , Erickson‐Miller Connie , Kikly Kristine , Camilleri Patrick
Published: March 2003

The siglecs (sialic acid‐binding immunoglobulin‐like lectins) mediate sialic acid‐dependent cellular interactions and may in some cases signal through SH2‐binding domains. In addition to the previously characterized siglecs, sialoadhesin, CD22, CD33 and myelin‐associated glycoprotein, several new ones, siglec‐5, siglec‐7 and siglec‐8, have recently been cloned. Although these novel receptors have generated considerable interest as therapeutic targets because of their expression pattern on immune cells, very little is known about how their lectin activity is regulated. Previous studies with sialoadhesin, CD22 and CD33 have shown that siglec glycosylation has significant effects on binding. To determine any differences in the glycan composition of siglec‐5, siglec‐7 and siglec‐8 that may modify their function, we released and characterized the N‐linked oligosaccharide distribution in these three glycoproteins. The glycan pools from siglec‐5 and siglec‐7 contained a larger proportion of sialylated and core‐fucosylated biantennary, triantennary and tetra‐antennary oligosaccharides, whereas the carbohydrate mixture released from siglec‐8 is noticeably less sialylated and is more abundant in ‘high‐mannose’‐type glycans. In addition, we show that, in contrast with CD22 and CD33, mutating the conserved potentially N‐linked glycosylation site in the first domain has no effect on binding mediated by siglec‐5 or siglec‐7.
Identification and characterization of SEB, a novel protein that binds to the acute undifferentiated leukemia‐associated protein SET
Authors: Minakuchi Masayoshi , Kakazu Naoki , Gorrin‐Rivas Manuel J. , Abe Tatsuo , Copeland Terry D. , Ueda Kunihiro , Adachi Yoshifumi
Published: March 2003

SET, the translocation breakpoint‐encoded protein in acute undifferentiated leukemia (AUL), is a 39‐kDa nuclear phosphoprotein and has an inhibitory activity for protein phosphatase 2A (PP2A). SET is fused to a putative oncoprotein, CAN/NUP214, in AUL and is thought to play a key role in leukemogenesis by its nuclear localization, protein–protein interactions and PP2A inhibitory activity. Here, we describe the isolation and characterization of a novel cDNA encoding a protein with 1542 amino‐acid residues that specifically interacts in a yeast two‐hybrid system as well as in human cells with SET. This new protein, which we name SEB (SET‐binding protein), is identified as a 170‐kDa protein by immunoprecipitation with a specific antibody and is localized predominantly in the nucleus. SEB1238–1434 is determined as a SET‐binding region that specifically binds to SET182–223. SEB also has an oncoprotein Ski homologous region (amino acids 654–858), six PEST sequences and three sequential PPLPPPPP repeats at the C‐terminus. SEB mRNA is expressed ubiquitously in all human adult tissues and cells examined. The SEB gene locus is assigned to the chromosome 18q21.1 that contains candidate tumor suppressor genes associated with deletions in cancer and leukemia. Although the function of SEB is not known, we propose that SEB plays a key role in the mechanism of SET‐related leukemogenesis and tumorigenesis, perhaps by suppressing SET function or by regulating the transforming activity of Ski in the nucleus.
Biosynthesis of cannabinoids
Authors: Fellermeier Monika , Eisenreich Wolfgang , Bacher Adelbert , Zenk Meinhart H.
Published: March 2003

The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1‐13C]glucose or [U‐13C6]glucose. 13C‐labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. 13C enrichments and coupling patterns show that the C10‐terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide‐type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.
Cyclic dipeptide oxidase from Streptomyces noursei
Authors: Gondry Muriel , Lautru Sylvie , Fusai Guillaume , Meunier Gilles , Ménez André , Genet Roger
Published: March 2003

Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of α,β‐dehydro‐Phe (ΔPhe) and α,β‐dehydro‐Leu (ΔLeu) residues during the biosynthesis of albonoursin, cyclo(ΔPhe‐ΔLeu), an antibiotic produced by Streptomyces noursei. It was purified 600‐fold with a 30% overall recovery, and consists of the association of a single type of subunit with a relative molecular mass of 21 066 resulting in a large homopolymer of relative molecular mass over 2 000 000. The enzyme exhibits a typical flavoprotein spectrum with maxima at 343.5 and 447.5 nm, the flavin prosthetic group being covalently bound to the protein. The catalytic reaction of the natural substrate cyclo(l‐Phe‐l‐Leu) occurs in a two‐step sequential reaction leading first to cyclo(α,β‐dehydro‐Phe‐l‐Leu) and finally to albonoursin. Kinetic parameters for the first step were determined (Km = 53 µm; k = 0.69 s−1). The enzyme was shown to catalyze the conversion of a variety of cyclo(dipeptides) and can be reoxidized at the expense of molecular oxygen by producing H2O2. This reaction mechanism, which differs from those already described for the formation of α,β‐dehydro‐amino acids, might consist of the transient formation of an intermediate imine followed by its rearrangement into an α,β‐dehydro‐residue.
Expression of cardiotoxin‐2 gene
Authors: Ma Donghui , Armugam Arunmozhiarasi , Jeyaseelan Kandiah
Published: March 2003

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)‐2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500‐bp (spliced) CTX‐2 mRNA as the final transcript. However, the liver produces two types of CTX‐2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP‐1, C/EBP, CACCC‐binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.
The structure of the PII–ATP complex
Authors: Xu Yibin , Carr Paul D. , Huber Thomas , Vasudevan Subhash G. , Ollis David L.
Published: March 2003

PII is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of PII was solved using a hexagonal crystal form (form I). The more physiologically relevant form of PII is a complex with small molecule effectors. We describe the structure of PII with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co‐crystallization of PII with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the PII/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that PII and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.
A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids
Authors: Pichler Harald , Gaigg Barbara , Hrastnik Claudia , Achleitner Georg , Kohlwein Sepp D. , Zellnig Günther , Perktold Andreas , Daum Günther
Published: March 2003

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta1234, 214–220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several‐fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and sterolδ24‐methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.
Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry
Authors: Garnier Cyrille , Lafitte Daniel , Jorgensen Thomas J. D. , Jensen Ole N. , Briand Claudette , Peyrot Vincent
Published: March 2003

HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin‐microfilament, tubulin‐microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms α‐HSP90 and β‐HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI‐MS and MALDI‐MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI‐MS, the α isoform being ≈ six times more abundant than the β isoform. ESI‐MS in combination with λ phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain α‐HSP90, with the diphosphorylated form being the most abundant. For the β isoform, the di‐phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross‐linking showed a high percentage of α–α homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.
Self‐assembled pearling structure of long duplex DNA with histone H1
Authors: Yoshikawa Yuko , Velichko Yuri S. , Ichiba Yuta , Yoshikawa Kenichi
Published: March 2003

We report that complexes of giant DNA molecules with histone H1 proteins form a pearl necklace‐like structure when the complexes are prepared by natural dilution from a high‐salt solution (2 m NaCl) to a low‐salt solution (0.2 m and 50 mm NaCl). We performed real‐time observations on the conformational changes of individual T4 phage DNA (166 kb) molecules in bulk solution by fluorescence microscopy. To identify H1‐binding regions on individual DNA molecules, we also performed immunofluorescence microscopic observations on the DNA–H1 complex spread on a glass surface. It was found that histone H1 binds DNA in a highly co‐operative manner and is accompanied by local folding of the DNA. On the basis of the experimental observations and a theoretical simulation, we propose a self‐assembling mechanism for the pearling structure.
Cloning and characterization of a novel serine/threonine protein kinase gene expressed predominantly in developing brain
Authors: Nara Kiyomitsu , Akasako Yuko , Matsuda Yoichi , Fukazawa Yugo , Iwashita Shintaro , Kataoka Masakazu , Nagai Yoshitaka
Published: March 2003

We have isolated a rat gene, sbk, that encodes a novel serine/threonine protein kinase possessing a consensus sequence for an SH3‐binding domain from developing rat brain. Rat SBK comprises 417 amino‐acid residues consisting of a serine/threonine protein kinase consensus sequence followed by a C‐terminal proline‐rich region. Sequence comparison with other known kinases revealed that sbk belongs to a novel family of serine/threonine protein kinases structurally related to a Xenopus gastrula‐specific protein kinase, Pk9.7. An in vitro kinase assay demonstrated that the SBK protein autophosphorylates at serine/threonine residues. Transcripts of sbk were strongly detected in brain, and the distribution shows an association with neurons but not glial cells. A marked increase in sbk transcripts was observed in developing brain in the late embryonic stage when dramatic neuronal proliferation, migration, and maturation occur. Fluorescence in situ hybridization analysis was used to map sbk to mouse chromosome 7F1‐F3 and rat chromosome 1q21. These data suggest a role for SBK in signal‐transduction pathways related to the control of brain development.
Cloning and characterization of the cDNA and gene for human epitheliasin
Authors: Jacquinet Eric , Rao Narayanam V. , Rao Gopna V. , Zhengming Wang , Albertine Kurt H. , Hoidal John R.
Published: March 2003

Previously, we reported cloning and characterization of the mouse gene, epitheliasin. In the present work we cloned the cDNA of the full‐length human orthologue and characterized its gene including 2 kb of 5′ flanking sequence. Analysis of epitheliasin gene expression in adult tissues shows that it is expressed as 3.4 kb and 2 kb transcripts. The major 3.4 kb transcript is observed in the following order: prostate > colon > small intestine > pancreas > kidney > lung > liver. Epitheliasin transcripts in fetal tissues are observed only in kidney and lung. In situ hybridization analysis of tissues revealed that epitheliasin was preferentially expressed in epithelial cells. The gene consists of 14 exons and 13 introns based on comparison with its cDNA sequence. In the 5′ flanking region, we identified two transcription start sites and three CpG islands encompassing a number of potential regulatory elements including SP1, SREBP, GRE/PRE and ERE. The region upstream of the transcription sites lacks a TATA box but contains an initiator‐like element as well as a downstream promoter‐like element. In vitro experiments with lymph node carcinoma of prostate (LNCaP) cells revealed that the epitheliasin gene was induced by androgens and the induction was not blocked by cycloheximide indicating that the induction required no intermediate protein factors. Immunoprecipitation analysis showed that androgens strongly increased epitheliasin protein levels.
N‐Terminally extended human ubiquitin‐conjugating enzymes (E2s) mediate the ubiquitination of RING‐finger proteins, ARA54 and RNF8
Authors: Ito Kenichi , Adachi Seiji , Iwakami Reiko , Yasuda Hideyo , Muto Yoshinori , Seki Naohiko , Okano Yukio
Published: March 2003

We have previously cloned cDNAs encoding the N‐terminally extended class III human ubiquitin‐conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biological functions of which are not known. In this study, we performed yeast two‐hybrid screening for protein(s) interacting with UBE2E2, and two RING‐finger proteins, ARA54 and RNF8, were identified. Both ARA54, a ligand‐dependent androgen receptor coactivator, and RNF8 interacted with class III E2s (UBE2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCdc34, and hBendless) in the yeast two‐hybrid assay. The use of various deletion mutants of UBE2E2 and RING‐finger proteins and two RING point mutants, ARA54 C(220)S and RNF8 C(403)S, in which the RING structure is disrupted, showed that the UBC domain of UBE2E2 and the RING domain of these RING‐finger proteins were involved in this association. Wild‐type ARA54 and RNF8, expressed in insect Sf9 cells, catalyzed E2‐dependent autoubiquitination in vitro, whereas the point mutated proteins showed markedly reduced activity. Ubiquitination of wild‐type ARA54 and RNF8, expressed in COS‐7 cells, was also observed, and a proteasome inhibitor, MG132, prevented the degradation of these wild‐type proteins, but was much less effective in protecting the RING mutants. Transfection of COS‐7 cells with a green fluorescent protein chimera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus. Our results suggest that ARA54 and RNF8 possibly act as Ub‐ligases (E3) in the ubiquitination of certain nuclear protein(s).
Biosynthesis of terpenoids
Authors: Rohdich Felix , Eisenreich Wolfgang , Wungsintaweekul Juraithip , Hecht Stefan , Schuhr Christoph A. , Bacher Adelbert
Published: March 2003

The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate synthase was expressed in a recombinant E. coli strain. The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate from 4‐diphosphocytidyl‐2C‐methyl‐d‐erythritol 2‐phosphate at a rate of 4.3 µmol·mg−1·min−1. At lower rates, the recombinant protein catalyzes the formation of 2‐phospho‐2C‐methyl‐d‐erythritol 3,4‐cyclophosphate from 4‐diphosphocytidyl‐2C‐methyl‐d‐erythritol 2‐phosphate and the formation of 2C‐methyl‐d‐erythritol 3,4‐cyclophosphate from 4‐diphosphocytidyl‐2C‐methyl‐d‐erythritol. Divalent metal ions such as magnesium or manganese are required for catalytic activity. The enzyme has a pH optimum at pH 7.0. Recombinant expression of the full‐length open reading frame afforded insoluble protein that could not be folded in vitro. The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis.
Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase
Authors: Cho Sung‐Woo , Yoon Hye‐Young , Ahn Jee‐Yin , Lee Eun‐Young , Lee Jongweon
Published: March 2003

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff’s base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2‐oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5′‐phosphate (PLP). For these studies, a 1557‐bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild‐type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400‐fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2‐oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild‐type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.
The pro‐sequence facilitates folding of human nerve growth factor fromEscherichia coliinclusion bodies
Authors: Rattenholl Anke , Lilie Hauke , Grossmann Adelbert , Stern Anne , Schwarz Elisabeth , Rudolph Rainer
Published: March 2003

Nerve growth factor (β‐NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro‐proteinin vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro‐peptide is so far unknown. As the pro‐sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human β‐NGF pro‐protein was compared to that of the mature form. Expression of rh‐pro‐NGF inEscherichia coliled to the formation of inclusion bodies (IBs). The presence of the covalently attached pro‐sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to thein vitrorefolding of mature NGF (rh‐NGF). Physicochemical characterization revealed that rh‐pro‐NGF is a dimer. The pro‐peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh‐NGF. Furthermore, rh‐pro‐NGF exhibited biological activity in the same concentration range as rh‐NGF.
Catalytic selenols couple the redox cycles of metallothionein and glutathione
Authors: Chen Yu , Maret Wolfgang
Published: March 2003

Co‐ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidation of its ligand. This molecular property links the binding and release of zinc in metallothionein (MT) to the cellular redox state [Maret W. & Vallee B.L. (1998) Proc. Natl Acad. Sci. USA95, 3483–3488]. Biological disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concentrations of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compound that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidation and reduction. Remarkably, selenium compounds catalyze the oxidation of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc–thiolate co‐ordination sites is linked to redox catalysis by selenium compounds, changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacological actions of selenium compounds in cancer prevention and other antiviral and anti‐inflammatory therapeutic applications, as well as unknown functions of selenium‐containing proteins, may relate to coupling between the thiol redox state and the zinc state.
Benzalacetone synthase
Authors: Abe Ikuro , Takahashi Yusuke , Morita Hiroyuki , Noguchi Hiroshi
Published: March 2003

Benzalacetone synthase (BSA) is a novel plant‐specific polyketide synthase that catalyzes a one step decarboxylative condensation of 4‐coumaroyl‐CoA with malonyl‐CoA to produce the C6–C4 skeleton of phenylbutanoids in higher plants. A cDNA encoding BAS was for the first time cloned and sequenced from rhubarb (Rheum palmatum), a medicinal plant rich in phenylbutanoids including pharmaceutically important phenylbutanone glucoside, lindleyin. The cDNA encoded a 42‐kDa protein that shares 60–75% amino‐acid sequence identity with other members of the CHS‐superfamily enzymes. Interestingly, R. palmatum BAS lacks the active‐site Phe215 residue (numbering in CHS) which has been proposed to help orient substrates and intermediates during the sequential condensation of 4‐coumaroyl‐CoA with malonyl‐CoA in CHS. On the other hand, the catalytic cysteine‐histidine dyad (Cys164–His303) in CHS is well conserved in BAS. A recombinant enzyme expressed in Escherichia coli efficiently afforded benzalacetone as a single product from 4‐coumaroyl‐CoA and malonyl‐CoA. Further, in contrast with CHS that showed broad substrate specificity toward aliphatic CoA esters, BAS did not accept hexanoyl‐CoA, isobutyryl‐CoA, isovaleryl‐CoA, and acetyl‐CoA as a substrate. Finally, besides the phenylbutanones in rhubarb, BAS has been proposed to play a crucial role for the construction of the C6–C4 moiety of a variety of natural products such as medicinally important gingerols in ginger plant.
The drug/metabolite transporter superfamily
Authors: Jack Donald L. , Yang Nelson M. , H. Saier Milton
Published: March 2003

Previous work defined several families of secondary active transporters, including the prokaryotic small multidrug resistance (SMR) and rhamnose transporter (RhaT) families as well as the eukaryotic organellar triose phosphate transporter (TPT) and nucleotide‐sugar transporter (NST) families. We show that these families as well as several other previously unrecognized families of established or putative secondary active transporters comprise a large ubiquitous superfamily found in bacteria, archaea and eukaryotes. We have designated it the drug/metabolite transporter (DMT) superfamily (transporter classification number 2.A.7) and have shown that it consists of 14 phylogenetic families, five of which include no functionally well‐characterized members. The largest family in the DMT superfamily, the drug/metabolite exporter (DME) family, consists of over 100 sequenced members, several of which have been implicated in metabolite export. Each DMT family consists of proteins with a distinctive topology: four, five, nine or 10 putative transmembrane α helical spanners (TMSs) per polypeptide chain. The five TMS proteins include an N‐terminal TMS lacking the four TMS proteins. The full‐length proteins of 10 putative TMSs apparently arose by intragenic duplication of an element encoding a primordial five‐TMS polypeptide. Sequenced members of the 14 families are tabulated and phylogenetic trees for all the families are presented. Sequence and topological analyses allow structural and functional predictions.