Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Primary structure of cytochrome c′ of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c′-type cytochromes

Cytochrome c′ of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome c ₅₅₅. This cytochrome is spectrally similar to other cytochromes c′ but is larger (16,000 Da) and has a lower midpoint potential (–205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c′ from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c′, only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c′ from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c′-type cytochrome which developed a covalent cross-link between a lysine residue and the c′-heme. Received: 26 May 1999 / Accepted: 9 September 1999     

Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket

The α2 isoform of Na,K-ATPase plays a crucial role in Ca²⁺ handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1). We have examined an hypothesis that instability of α2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to α2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in α1, singly or together. Judged by the sensitivity of the purified proteins to heat, detergent, “affinity” for phosphatidylserine, and stabilization by FXYD1, the triple mutant (A920V/L955F/V981P, called α2VFP) has stability properties close to α1, although single mutants have only modest or insignificant effects. Functional differences between α1 and α2 are unaffected in α2VFP. A compound, 6-pentyl-2-pyrone, isolated from the marine fungus Trichoderma gamsii is a novel probe of specific phospholipid-protein interactions. 6-Pentyl-2-pyrone inactivates the isoforms in the order α2 ≫ α3 > α1, and α2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain α1, α2, and α3 isoforms, a component attributable to α2 is the least stable. The data provide clear evidence for a specific phosphatidylserine binding pocket between M8, M9, and M10 and confirm that the instability of α2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of α2 may be important for its cellular regulatory functions.     

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t_(1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol⁻¹ for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.     

Stabilization of RNA Bulges by Oligonucleotides Containing 2′-Naphthylmethyl-2′-deoxytubercidine

Abstract

A novel nucleoside analogue, 2′-naphthylmethyl-2′-deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.     

Stabilization of the μ-Opioid Receptor by Truncated Single Transmembrane Splice Variants through a Chaperone-like Action

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5′ and 3′ alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

The oxidative nuclease activity of human cytochrome c with mutations in Ω-loop C/D

Natural and artificial nucleases have extensive applications in biotechnology and biomedicine. The exploration of protein with potential DNA cleavage activity also inspires the design of artificial nuclease and helps to understand the physiological process of DNA damage. In this study, we engineered four human cytochrome c (Cyt c) mutants (N52S, N52A, I81N, and I81D Cyt c), which showed enhanced DNA cleavage activity and degradation in comparison with WT Cyt c, especially under acidic conditions. The mechanism assays revealed that the superoxide (O₂^??) plays an important role in the nuclease reaction. The kinetic assays showed that the peroxidase activity of the I81D Cyt c mutant enhanced up to 9-fold at pH 5. This study suggests that the mutations of Ile81 and Asn52 in Ω-loop C/D are critical for the nuclease activity of Cyt c, which may have physiological significance in DNA damage and potential applications in biomedicine.     

Comparative hydrogen-deuterium exchange for a mesophilic vs thermophilic dihydrofolate reductase at 25 °C: identification of a single active site region with enhanced flexibility in the mesophilic protein

The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].     

Stabilization of Myc through Heterotypic Poly-Ubiquitination by mLANA Is Critical for γ-Herpesvirus Lymphoproliferation

Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC₅S^mLANA modulates cellular control of Myc turnover by antagonizing SCF^Fbw7 mediated proteasomal degradation of Myc, mimicking SCF^β-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.     

The novel cytochrome c6 of chloroplasts: a case of evolutionary bricolage?

Cytochrome c6 has long been known as a redox carrier of the thylakoid lumen of cyanobacteria and some eukaryotic algae that can substitute for plastocyanin in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However, a homologue of the protein has now been identified in several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This form of the protein, designated cytochrome c6A, differs from the 'conventional' cytochrome c6 in possessing a conserved insertion of 12 amino acids that includes two absolutely conserved cysteine residues. There are conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron transfer. The evidence for and against this is reviewed and the likely evolutionary history of cytochrome c6A is discussed. It is suggested that it has been converted from a primary role in electron transfer to one in regulation within the chloroplast, and is an example of evolutionary 'bricolage'.     

M?ssbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with M?ssbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with M?ssbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The M?ssbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.     

Mechanism of substrate hydrolysis by a thermophilic endoglucanase from Thermotoga maritima†

A cellulase from the thermophile, Thermotoga maritima, hydrolyzed oligosaccharide substrates by an exoglucanase mode of action but acted as an endoglucanase to rapidly reduce the viscosity of the soluble polysaccharides carboxymethylcellulose and barley -glucan. The V _max for hydrolysis of the substrate, p-nitrophenyl -d-cellobioside, was 42 mol min^–1 (mg protein)^–1, while that for barley -glucan was 637. The enzyme had little activity on crystalline cellulose.     

Bleaching of euglenas by antibiotics—a specific form of antagonism in actinomycetes

The author discusses little investigated antagonistic effects of certain antibiotics, manifested by the inhibition of chlorophyll synthesis inEuglena gracilis. Two main types of depigmentation were found—temporary and permanent. The oldest substrains ofEuglena gracilis var.bacillaris, strain Mainx, permanently bleached by erthromycin, streptomycin, carbomycin, spiramycin, kanamycin and viomycin, were kept in the light for at least two years without showing any signs of turning green. These antibiotics caused permanent apochlorosis, but reversible apoplastidy. The euglena cultures remained permanently heterotrophic, Picromycin, methymycin and neomycin. however, only temporarily inhibited chlorophyll synthesis. When transferred to medium not containing these antibiotics and incubated in the light, these cultures again produced chlorophyll.     

Cold adaptation of a mesophilic cellulase,EG Ⅲ from Trichoderma reesei,by directed evolution

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold-adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG Ⅲ with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG Ⅲ calculated from Arrhenius equation are 13.3 kJ· mol-1 and 26.2 kJ ·mol-1, respectively. Therefore, the increased specific activity of w-3 at lower tempera