Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.

Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the –1958 bp AS promoter linked to the β-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in –1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than –405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least –640 bp of the AS promoter but not those with –523 bp or smaller promoter fragments. Fusion of the –640 to –523 bp region to a –381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.

Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.

Evolution of resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum

The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum malaria. Although the molecular basis for parasite resistance is known, the factors promoting the development and transmission of these resistant parasites are less clear. This paper reports the results of a quantitative comparison of factors previously hypothesized as important for the development of drug resistance, drug dosage, time of treatment, and drug elimination half-life, with an in-host dynamics model of P. falciparum malaria in a malaria-naive host. The results indicate that the development of drug resistance can be categorized into three stages. The first is the selection of existing parasites with genetic mutations in the dihydrofolate reductase or dihydropteroate synthetase gene. This selection is driven by the long half-life of the sulfadoxine-pyrimethamine combination. The second stage involves the selection of parasites with allelic types of higher resistance within the host during an infection. The timing of treatment relative to initiation of a specific anti-P. falciparum EMP1 immune response is an important factor during this stage, as is the treatment dosage. During the third stage, clinical treatment failure becomes prevalent as the parasites develop sufficient resistance mutations to survive therapeutic doses of the drug combination. Therefore, the model output reaffirms the importance of correct treatment of confirmed malaria cases in slowing the development of parasite resistance to sulfadoxine-pyrimethamine.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Stable production of hyaluronic acid in Streptococcus zooppidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h(-1)) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci. (c) 2005 Wiley Periodicals, Inc.

The ABC transporter gene in the sirodesmin biosynthetic gene cluster of Leptosphaeria maculans is not essential for sirodesmin production but facilitates self-protection

Epipolythiodioxopiperazine toxins are secreted by a range of fungi, including Leptosphaeria maculans, which produces sirodesmin, and Aspergillus fumigatus, which produces gliotoxin. The L. maculans biosynthetic gene cluster for sirodesmin includes an ABC transporter gene, sirA. Disruption of this gene led to increased secretion of sirodesmin into the medium and an altered ratio of sirodesmin to its immediate precursor. The transcription pattern of a peptide synthetase that catalyses an early step in sirodesmin biosynthesis was elevated in the sirA mutant by 47% over a 7-day period. This was consistent with the finding that the transporter mutant had elevated sirodesmin levels. Despite increased production of sirodesmin, the sit-A mutant was more sensitive to both sirodesmin and gliotoxin. The putative gliotoxin transporter gene, gliA, (a major facilitator superfamily transporter) from A.fumigatus complemented the tolerance of the L. maculans sirA mutant to gliotoxin, but not to sirodesmin. The results indicate that SirA contributes to self-protection against sirodesmin in L. maculans and suggest a transporter other than SirA is primarily responsible for efflux of endogenously produced sirodesmin. (C) 2004 Elsevier Inc. All rights reserved.