The phylogenetic origin of the bifunctional tyrosine-pathway protein in the enteric lineage of bacteria

Because bifunctional enzymes are distinctive and highly conserved products of relatively infrequent gene-fusion events, they are particularly useful markers to identify clusters of organisms at different hierarchical levels of a phylogenetic tree. Within the subdivision of gram-negative bacteria known as superfamily B, there are two distinctive types of tyrosine-pathway dehydrogenases: (1) a broad- specificity dehydrogenase (recently termed cyclohexadienyl dehydrogenase [CDH]) that can utilize either prephenate or L-arogenate as alternative substrates and (2) a bifunctional CDH that also posseses chorismate mutase activity. (T-proteins). The bifunctional T-protein, thought to be encoded by fused ancestral genes for chorismate mutase and CDH, was found to be present in enteric bacteria (Escherichia, Shigella, Salmonella, Citrobacter, Klebsiella, Erwinia, Serratia, Morganella, Cedecea, Kluyvera, Hafnia, Edwardsiella, Yersinia, and Proteus) and in Aeromonas and Alteromonas. Outside of the latter "enteric lineage," the T-protein is absent in other major superfamily-B genera, such as Pseudomonas (rRNA homology group I), Xanthomonas, Acinetobacter, and Oceanospirillum. Hence, the T-protein must have evolved after the divergence of the enteric and Oceanospirillum lineages. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase-phe, an early-pathway isozyme sensitive to feedback inhibition by L- phenylalanine, has been found in each member of the enteric lineage examined. The absence of both the T-protein and DAHP synthase-phe elsewhere in superfamily B indicates the emergence of these character states at approximately the same evolutionary time.      

Characterization of the F plasmid bifunctional conjugation gene, traG.

Summary The Escherichia coli F plasmid gene, traG, is required for two stages of the conjugation process: pilus biosynthesis and mating aggregate stabilization. The nucleotide sequence of traG has been determined and the topology of its product in the cytoplasmic membrane analysed using protease accessibility experiments. Complementation analysis employing plasmid deletions revealed a correlation between an N-terminal periplasmic segment of the protein product (TraGp) and its pilus assembly activity. Production of an anti-TraGp antiserum has facilitated the detection of TraGp^*, a possible internal cleavage product of TraGp. Although its function is unknown, TraGp^* is located in the periplasm and has been shown to possess sequences required for aggregate stabilization. The detection of TraGp^*raises the possibility that the two functions of traG are carried out by separate products.     

The recent evolutionary origin of the phenylalanine-sensitive isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in the enteric lineage of bacteria

Summary Evolutionary events that generated the three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase present in contemporary strains ofEscherichia coli have been proposed recently [Ahmad et al. (1986) J Bacteriol 165:146–154]. The phylogenetic subdivision of gram-negative prokaryotes studied (Superfamily B) includes enteric bacteria, anOceanospirillum cluster, pseudomonad Group I (e.g.,Pseudomonas aeruginosa), pseudomonad Group V (e.g.,Xanthomonas), and theAcinetobacter grouping. DAHP synthase-phe, a regulatory isozyme subject to allosteric control byl-phenylalanine, was the last member of the isozyme family to evolve. Thus, DAHP synthase-phe is absent throughout Superfamily B except within the enteric lineage. Bacteria that make up the enteric lineage (Escherichia, Klebsiella, Erwinia, Serratia, Proteus, Aeromonas, andAlteromonas) were examined in detail; DAHP synthasephe was present in each of these organisms. Therefore, the isozyme originated between the separation of the enteric andOceanospirillum lineages, prior to the divergence ofAlteromonas putrefaciens (44% homology withE. coli by DNA:rRNA hybridization) from the rest of the enteric lineage. DAHP synthase-tyr and DAHP synthase-trp were uniformly present within the enteric lineage, although it was often necessary to derepress DAHP synthase-trp by physiological manipulation in order to demonstrate its presence.     

Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein

Recombinant Saccharomyces cerevisiae YPB-G strain secreting a fusion protein displaying both BsAAase/GAase activities was grown in 1.5 l YPS media containing single (starch) and mixed carbon sources (glucose+starch) using a 2.5 l New Brunswick BiofloIII fermenter. Ethanol and biomass formation, starch utilisation, secretion of the amylolytic enzymes (-amylase and glucoamylase), accumulation of reducing sugars and glucose were followed during the fermentation of YPB-G under different conditions. Moreover, a model has been developed for the growth of recombinant yeast on substitutable substrates using cybernetic framework principles and incorporating product formation. In the present work, both the biphasic and the diauxic growth patterns observed experimentally in batch culture of recombinant yeast cells were simulated successfully by modifying the cybernetic framework to include ethanol formation and the degradation kinetics of starch which is not directly utilised by yeast. The model can further be expanded to fed-batch systems.     

Phenotypic variability of beta-glucoside utilization and its correlation to pathogenesis process in a few enteric bacteria

Utilization of beta-glucosides is markedly variable in the members of the family Enterobacteriaceae. The results presented here provide molecular clues for evolutionary events that resulted in the phenotypic variability seen amongst the members of these species. The genomic hybridization of selected Enterobacteriaceae members with the Escherichia coli bgl and cel genes resulted in detection of a complete homolog of the bgl and cel operons in Shigella sonnei, a member that is evolutionarily closest to E. coli. However, the Salmonella group of organisms have been shown to carry only a homolog of bglR and bglG regions and the deletions of the bglF and bglB genes. Similarly, Proteus mirabilis, Enterobacter aerogenes and a non-enteric Gram-negative bacterium Pseudomonas aeruginosa have been shown to carry a homolog of the bglR and bglG regions and deletions of the bglF and bglB genes. The homolog of the cel operon could be identified in S. sonnei and Salmonella groups of organisms. Possible implications of these observations, in connection with the phenotypic variability seen in beta-glucoside utilization amongst these members, are discussed.     

Crystal structure of CelM2, a bifunctional glucanase-xylanase protein from a metagenome library

Degradation of polysaccharides by cellulases and xylanases plays an important role in the carbon cycle, but only occurs in plant cell walls, a few bacteria and some animals. This process is also critical in processes such as biomass degradation and fuel production in the conversion cycles of cellulosic biomass. The enzyme CelM2 is bifunctional, because it is able to effectively hydrolyze barley glucan and xylan. Here, we show the crystal structure of the bifunctional enzyme CelM2, isolated from a metagenome library, and describe the sequence information and structure of its two domains. We believe that CelM2 is attractive as an industrial enzyme and that the structural results presented herein provide insights that are relevant to the genetic engineering of multifunctional enzymes.     

A molecular phylogeny of enteric bacteria and implications for a bacterial species concept

A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.     

The prephenate dehydrogenase component of the bifunctional T-protein in enteric bacteria can utilize L-arogenate

The prephenate dehydrogenase component of the bifunctional T-protein (chorismate mutase:prephenate dehydrogenase) has been shown to utilize L-arogenate, a common precursor of phenylalanine and tyrosine in nature, as a substrate. Partially purified T-protein from Klebsiella pneumoniae and from Escherichia coli strains K 12, B, C and W was used to demonstrate the utilization of L-arogenate as an alternative substrate for prephenate in the presence of nicotinamide adenine dinucleotide as cofactor. The formation of L-tyrosine from L-arogenate by the T-protein dehydrogenase was confirmed by high-performance liquid chromatography. As expected of a common catalytic site, dehydrogenase activity with either prephenate or L-arogenate was highly sensitive to inhibition by L-tyrosine.     

Enhanced cytotoxic activity of a bifunctional chimeric protein containing a type 1 ribosome-inactivating protein and a serine protease inhibitor

Both ribosome-inactivating proteins (RIPs) and plant proteinase inhibitors, belong to protein families known to regulate cellular homeostasis and likely involved in plant defense. Nevertheless the interest in these protein classes is due to their potential use for the treatment of several important human diseases such as cancer. Thus, in the present study, type 1 ribosome-inactivating protein and wheat subtilisin/chymotrypsin inhibitor, were engineered into a chimeric protein with cytotoxic action selective for murine tumor cells, while lacking any appreciable toxicity on murine normal cells. This chimeric protein selectively sensitizes to apoptotic death cells derived from Simian-virus-40-transformed mouse fibroblasts (SVT2 cells). The cytotoxicity of this new recombinant product has been detected also on three different human malignant cells. Therefore action on tumor cells of this protein could represent a potentially very attractive novel tool for anticancer drug design.     

The fusion protein core of measles virus forms stable coiled-coil trimer

Recent studies have shown that paramyxovirus might adopt a similar molecular mechanism of virus entry and fusion in which the attachment glycoprotein binds receptor/s and triggers the conformational changes of the fusion protein. There are two conserved regions of heptad repeat (HR1 and HR2) in the fusion protein and they were shown with fusion-inhibition effects in many paramyxoviruses, including measles virus. They also appear to show characteristic structure in the fusion core: the HR1/HR2 forms stable six-helix coiled-coil centered by HR1 and is surrounded by HR2 (trimer of HR1/HR2), which represents the post-fusion conformational structure. In this study, we expressed the HR1 and HR2 of measles virus fusion protein as a single chain (named 2-Helix) and subsequently tested its formation of trimer. Indeed, the results do show that the HR1 and HR2 interact with each other and form stable six-helix coiled-coil bundle. This is the first member in genus Morbillivirus of family Paramyxoviridae to be confirmed with this characteristic structure and provides the basis for the HR2-inhibition effects on virus fusion/entry for measles virus.     

DisCons: a novel tool to quantify and classify evolutionary conservation of intrinsic protein disorder

Background

Analyzing the amino acid sequence of an intrinsically disordered protein (IDP) in an evolutionary context can yield novel insights on the functional role of disordered regions and sequence element(s). However, in the case of many IDPs, the lack of evolutionary conservation of the primary sequence can hamper the study of functionality, because the conservation of their disorder profile and ensuing function(s) may not appear in a traditional analysis of the evolutionary history of the protein.

Results

Here we present DisCons (Disorder Conservation), a novel pipelined tool that combines the quantification of sequence- and disorder conservation to classify disordered residue positions. According to this scheme, the most interesting categories (for functional purposes) are constrained disordered residues and flexible disordered residues. The former residues show conservation of both the sequence and the property of disorder and are associated mainly with specific binding functionalities (e.g., short, linear motifs, SLiMs), whereas the latter class correspond to segments where disorder as a feature is important for function as opposed to the identity of the underlying sequence (e.g., entropic chains and linkers). DisCons therefore helps with elucidating the function(s) arising from the disordered state by analyzing individual proteins as well as large-scale proteomics datasets.

Conclusions

DisCons is an openly accessible sequence analysis tool that identifies and highlights structurally disordered segments of proteins where the conformational flexibility is conserved across homologs, and therefore potentially functional. The tool is freely available both as a web application and as stand-alone source code hosted at http://pedb.vib.be/discons.     

Divergent evolution of a bifunctional de novo protein

Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn‐IF, was chosen from a library of binary patterned 4‐helix bundles. Syn‐IF was shown previously to rescue two different auxotrophic strains of E. coli: ΔilvA and Δfes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn‐IF, was evolved for faster rescue of either ΔilvA or Δfes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity.     

Concomitant selective adsorption and covalent immobilization of a His-tagged protein switch with silica-based metal chelate-epoxy bifunctional adsorbents

A series of silica-based bifunctional adsorbents containing both metal-chelating groups and epoxy groups for the concomitant purification and immobilization of His-tagged protein switch RG13, a potential bioreceptor for developing maltose biosensors, were prepared by controlling the ratio of iminodiacetic acid-conjugated silane (GLYMO-IDA) and silane (GLYMO) used for surface modification. The bifunctional adsorbent prepared with a [GLYMO-IDA]/[GLYMO] ratio of 0.2, containing a [metal chelating group]/[epoxy group] ratio of 1.42, was shown to exhibit a metal chelating capacity of 88.42 ± 15.91 μmole Cu²⁺/g, a protein adsorption capacity of 1.81 ± 0.19 mg/g and a superior selectivity over the other bifunctional adsorbents. Results of kinetic studies showed that selective adsorption and covalent bond formation at 4 °C were achieved in 1 h and 15 h, respectively, which allowed the sequential adsorption and covalent immobilization of protein switch RG13. A protein immobilization yield of 94.6 % and a global activity yield of 63.4 % were obtained, giving an immobilized protein switch RG13 with an enzymatic activity of 4.57 ± 0.19 U/g, under optimal conditions at pH 8.0 and 40 °C. In the repeated-batch operation, the bifunctional adsorbent-immobilized RG13 retained 91 % of the original activity after 20 cycles, 39 % higher than the counterpart prepared with monofunctional metal chelate adsorbent mediated solely by coordinate linkages.     

The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein

A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43 kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His₆ and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs’-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.     

Cloning of a beta-lactam resistance determinant of Enterobacter cloacae affecting outer membrane proteins of Enterobacteriaceae

Abstract In this paper we describe the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that causes β-lactam resistance in both Escherichia coli HB101 and the parental strain E. cloacae 2249-1.
The increase in minimum inhibitory concentration (MIC) of the β-lactam antibiotics studied was not the result of enhanced β-lactamase production, but of a decrease in the concentration of the pore proteins OmpF and OmpC in E. coli and of a 37-kDa membrane protein in E. cloacae . The results obtained thus far indicate that we have cloned a gene encoding a 20 kDa polypeptide that is involved in the regulation of outer membrane protein synthesis.     

基于新杂合进化算法的蛋白质折叠计算

蛋白质能量最小化是蛋白质折叠的重要内容。用于蛋白质折叠的新的杂合进化算法结合了交叉和柯西变异。基于toy模型的蛋白质能量最小化算例表明,这个新的杂合进化算法是有效的。     

The NMR structure of a stable and compact all-beta-sheet variant of intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) has a clam-shaped structure that may serve as a scaffold for the design of artificial enzymes and drug carriers. In an attempt to optimize the scaffold for increased access to the interior-binding cavity, several helix-less variants of I-FABP have been engineered. The solution-state NMR structure of the first generation helix-less variant, known as Delta17-SG, revealed a larger-than-expected and structurally ill-defined loop flanking the deletion site. We hypothesized that the presence of this loop, on balance, was energetically unfavorable for the stability of the protein. The structure exhibited no favorable pairwise or nonpolar interactions in the loop that could offset the loss of configurational entropy associated with the folding of this region of the protein. As an attempt to generate a more stable protein, we engineered a second-generation helix-less variant of I-FABP (Delta27-GG) by deleting 27 contiguous residues of the wild-type protein and replacing them with a G-G linker. The deletion site of this variant (D9 through N35) includes the 10 residues spanning the unstructured loop of Delta17-SG. Chemical denaturation experiments using steady-state fluorescence spectroscopy showed that the second-generation helix-less variant is energetically more stable than Delta17-SG. The three-dimensional structure of apo-Delta27-GG was solved using triple-resonance NMR spectroscopy along with the structure calculation and refinement protocols contained in the program package ARIA/CNS. In spite of the deletion of 27 residues, the structure assumes a compact all-beta-sheet fold with no unstructured loops and open access to the interior cavity.     

Glutamate-stimulated activation of DNA synthesis via mitogen-activated protein kinase in primary astrocytes: involvement of protein kinase C and related adhesion focal tyrosine kinase

Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time- and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate-dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.