Role of different β-turns in β-hairpin conformation and stability studied by optical spectroscopy

Model β-hairpin peptides based on variations in the turn sequence of Cochran's tryptophan zipper peptide, SWTWENGKWTWK, were studied using electronic circular dichroism (ECD), fluorescence, and infrared (IR) spectroscopies. The trpzip2 Asn-Gly turn sequence was substituted with Thr-Gly, Aib-Gly, (D)Pro-Gly, and Gly-Asn (trpzip1) to study the impact of turn stability on β-hairpin formation. Stability and conformational changes of these hairpins were monitored by thermodynamic analyses of the temperature variation of both FTIR (amide I') and ECD spectral intensities. These changes were fit to a two-state model which yielded different T(m) values, representing the folding/unfolding process, for hairpins with different β-turns. Different β-turns show systematic contributions to hairpin structure formation, and their inclusion in hairpin design can modify the folding pathways. Aib-Gly or (D)Pro-Gly sequences stabilize the turn resulting in residual Trp-Trp interaction at high temperatures, but at the same time the β-structure (cross strand H-bonds) can become less stable due to constraints of the turn, as seen for (D)Pro-Gly. The structure of the Aib-Gly turn containing hairpin was determined by NMR and was shown to be like trpzip2 (Asn-Gly turn) as regards turn and strand geometries, but to differ from trpzip1 (Gly-Asn turn). The Munoz and Eaton statistical mechanically derived multistate model, tested as an alternate point of view, represented contributions from H-bonds and hydrophobic interactions as well as conformational change as interdependent. Use of different spectral methods that vary in dependence on these physical interactions along with the structural variations provided insight to the complex folding pathways of these small, well-folded peptides.     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts     

Role of phages in the pathogenesis of Burkholderia,or ‘Where are the toxin genes in Burkholderia phages?’

Most bacteria of the genus Burkholderia are soil- and rhizosphere-associated, and rhizosphere associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens, and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens; however, the data do not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a 'versaphile' such that selection is occurring in several niches, including as a pathogen and in the context of environmental survival.     

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H₂O₂ (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillin N acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillin N) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.     

Role of carotenoid β-cryptoxanthin in bone homeostasis

Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Aging induces bone loss due to decreased osteoblastic bone formation and increased osteoclastic bone resorption. Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. Nutritional factors may play a role in the prevention of bone loss with aging. Among various carotenoids (carotene and xanthophylls including beta (β)-cryptoxanthin, lutein, lycopene, β-carotene, astaxanthin, and rutin), β-cryptoxanthin, which is abundant in Satsuma mandarin orange (Citrus unshiu MARC.), has been found to have a stimulatory effect on bone calcification in vitro. β-cryptoxanthin has stimulatory effects on osteoblastic bone formation and inhibitory effects on osteoclastic bone resorption in vitro, thereby increasing bone mass. β-cryptoxanthin has an effect on the gene expression of various proteins that are related osteoblastic bone formation and osteoclastic bone resororption in vitro. The intake of β-cryptoxanthin may have a preventive effect on bone loss in animal models for osteoporosis and in healthy human or postmenopausal women. Epidemiological studies suggest a potential role of β-cryptoxanthin as a sustainable nutritional approach to improving bone health of human subjects. β-Cryptoxanthin may be an osteogenic factor in preventing osteoporosis in human subjects.     

The Role of β-Hydroxypropionate in Ethylene Biosynthesis

To search precursors of ethylene in banana fruits, ethylene formation from acetate-2-¹⁴C and fumarate-2,3-¹⁴C by banana slices was studied. Ethylene-¹⁴C formation from acetate-2-^l4C was reduced by the addition of malonate or β-hydroxypropionate, and it was enhanced in a sealed chamber in comparison with the case in an aeration chamber. No label of fumarate-2,3-¹⁴C was incorporated into ethylene.

From these facts it was suggested that acetate-2-¹⁴C was incorporated into ethylene via malonate and β-hydroxypropionate. Participation of fumarate in ethylene biosynthesis of banana fruits was ruled out. β-Hydroxypropionate was postulated as an effective precursor of ethylene formation from acetate-2-^l4C.     

Soil Type and Maize Cultivar Affect the Genetic Diversity of Maize Root–Associated Burkholderia cepacia Populations

Abstract Burkholderia cepacia populations associated with the Zea mays root system were investigated to assess the influence of soil type, maize cultivar, and root localization on the degree of their genetic diversity. A total of 180 B. cepacia isolates were identified by restriction analysis of the amplified 16S rDNA (ARDRA technique). The genetic diversity among B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique, using the 10-mer primer AP5. The analysis of molecular variance (AMOVA) method was applied to estimate the variance components for the RAPD patterns. The results indicated that, among the factors studied, the soil was clearly the dominant one in affecting the genetic diversity of maize root–associated B. cepacia populations. In fact, the percentage of variation among populations was significantly higher between B. cepacia populations recovered from maize planted in different soils than between B. cepacia populations isolated from different maize cultivars and from distinct root compartments such as rhizoplane and rhizosphere. The analysis of the genetic relationships among B. cepacia isolates resulted in dendrograms showing bacterial populations with frequent recombinations and a nonclonal genetic structure. The dendrograms were also in agreement with the AMOVA results. We were able to group strains obtained from distinct soils on the basis of their origin, confirming that soil type had the major effect on the degree of genetic diversity of the maize root–associated B. cepacia populations analyzed. On the other hand, strains isolated from distinct root compartments exhibited a random distribution which confirmed that the rhizosphere and rhizoplane populations analyzed did not significantly differ in their genetic structure. Received: 22 January 1999; Accepted: 7 April 1999     

Role of α-phosphoglucomutase and phosphoglucose isomerase activities at the branching point between sugar catabolism and anabolism in Lactobacillus casei

Aims: To evaluate the role of α‐phosphoglucomutase (α‐Pgm) and phosphoglucose isomerase (Pgi) activities in growth rate, sugar‐phosphates, UDP‐sugars and lactate biosynthesis in Lactobacillus casei. Methods and Results: The pgm and pgi genes coding for α‐Pgm and Pgi activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous overexpression. In MRS fermentation medium with glucose, overexpression of pgm gene in L. casei resulted in a growth rate reduced to 75% and glucose‐6P levels reduced to 47%. By contrast, with lactose, the growth rate was raised to 119%. An increment of α‐Pgm activity had no significant effect on UDP‐sugar levels. Remarkably, Pgi overexpression in L. casei grown in lactose or galactose resulted in almost a double growth rate with respect to the control strain. The increased Pgi activity also resulted in glucose‐6P levels reduced to 25 and 59% of control strain cultured in glucose and lactose, respectively, and the fructose‐6P levels were increased to 128% on glucose. UDP‐glucose and UDP‐galactose levels were reduced to 66 and 55%, respectively, of control strain levels cultured in galactose. In addition, the lactate yield increased to 115% in the strain overproducing Pgi grown in galactose. Conclusions: The physiological amount of α‐Pgm and Pgi activities is limited for L. casei growth on lactose, and lactose and galactose, respectively, and that limitation was overcome by pgm and pgi gene overexpression. The increment of α‐Pgm and Pgi activities, respectively, resulted in modified levels of sugar‐phosphates, sugar‐nucleotides and lactate showing the modulation capacity of the carbon fluxes in L. casei at the level of the glycolytic intermediate glucose‐6P. Significance and Impact of the Study: Knowledge of the role of key enzymes in metabolic fluxes at the branching point between anabolic and catabolic pathways would allow a rational design of engineering strategies in L. casei.     

Alterations in the extracellular catabolism of nucleotides and platelet aggregation induced by high-fat diet in rats: effects of α-tocopherol

The aim of this study was to assess whether α-tocopherol administration prevented alterations in the ectonucleotidase activities and platelet aggregation induced by high-fat diet in rats. Thus, we examined four groups of male rats which received standard diet, high-fat diet (HFD), α-tocopherol (α-Toc), and high-fat diet plus α-tocopherol. HFD was administered ad libitum and α-Toc by gavage using a dose of 50 mg/kg. After 3 months of treatment, animals were submitted to euthanasia, and blood samples were collected for biochemical assays. Results demonstrate that NTPDase, ectonucleotide pyrophosphatase/phosphodiesterase, and 5′-nucleotidase activities were significantly decreased in platelets of HFD group, while that adenosine deaminase (ADA) activity was significantly increased in this group in comparison to the other groups (P?<?0.05). When rats that received HFD were treated with α-Toc, the activities of these enzymes were similar to the control, but ADA activity was significantly increased in relation to the control and α-Toc group (P?<?0.05). HFD group showed an increased in platelet aggregation in comparison to the other groups, and treatment with α-Toc significantly reduced platelet aggregation in this group. These findings demonstrated that HFD alters platelet aggregation and purinergic signaling in the platelets and that treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition.     

Isolation ofEscherichia coli mutants with simultaneous hyperproduction of d-serine deaminase and β-galactosidase

Summary Double mutants ofE. coli hyperproducing D-serine deaminase and -galactosidase were isolated by two successive selection procedures in the chemostat. The specific activity of D-serine deaminase is 10 times higher and -galactosidase 5 times higher compared with the fully induced original strain B 28.     

Role of Glu445 in the substrate binding of β-glucosidase

A previously uncharacterized gene in Neosartorya fischeri was cloned and expressed in Escherichia coli. It was found to encode a β-glucosidase (NfBGL1) distinguishable from other BGLs by its high turnover of p-nitrophenyl β-d-glucopyranoside (pNPG). Molecular determinants for the substrate recognition of NfBGL1 were studied through an initial screening of residues by sequence alignment, a second screening by homology modeling and subsequent site-directed mutagenesis to alter individual screened residues. A conserved amino acid, E445, in the substrate binding pocket of wild-type NfBGL1 was identified as an important residue affecting substrate affinity. Replacement of E445 with amino acids other than aspartate significantly decreased the catalytic efficiency (k_cat/K_m) of NfBGL1 towards pNPG, mainly through decreased binding affinity. This was likely due to the disruption of hydrogen bonding between the substrate and the carboxylate oxygen of the residue at position 445. Density functional theory (DFT) based studies suggested that an acidic amino acid at position 445 raises the substrate affinity of NfBGL1 through hydrogen bonding. The residue E445 is completely conserved indicating that this position can be considered as a crucial determinant for the substrate binding among GHs tested.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Production of β-fructofuranosidase by Aspergillus japonicus in batch and fed-batch cultures

Summary A comparison of -fructofuranosidase (FFase, EC 3.2.l.26) production by Aspergillus japonicus TIT-90076 in batch and fed-batch cultures was investigated in shaken flasks. Results showed that fed-batch cultivation of A. japonicus using intermittent sucrose supply produced more FFase than batch culture, and the maximal enzyme production was 910 units ml^–1, which was about 20% higher than that in the batch cultures.