A generalized theory of particulate electron conduction enzymes applied to cytochrome oxidase. A theory of coupled electron and/or ion transport applied to pyruvate carboxylase

A previously developed theory of particulate electron conduction enzymes was based on a model of an enzyme particle catalyzing the oxidation-reduction of two different substrates at two different enzymatic sites on the same particle with conduction of electrons between the two sites through the enzyme particle. Using the simplifying assumption that the percent reduction of the second substrate is held constant, there was previously shown to be a hyperbolic relationship between the first order rate constant (k′) and the sum (C _x ) of oxidized plus reduced substrate, of the formk′=α/(C _x +β), where α and β are positive constants. It is shown here that if this simplifying assumption is omitted, a positive constant is added to the right hand side of this equation, which describes exactly the experimental data of Smith and conrad on cytochrome oxidase. If electron transport is assumed to be coupled to ion transport, this equation becomesk′=(α/C _x )?γ (where γ is a positive constant) which describes the experimental data of Eadie and Gale on pyruvic carboxylase of yeast. It seems probable that the same theory is applicable to coupled ion-ion transport and coupled electron-electron transport in both membranous systems, and in particulate preparations consisting of membrane fragments.     

Catalytic properties of aminoacylase of strain <Emphasis Type="Italic">Rhodococcus armeniensis</Emphasis> AM6.1

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K_m) and maximum reaction velocities (V_max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K_s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K_s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K_i = 104.7 ± 21.7 mM, K_m = 2.5 ± 0.5 mM, V_max = 25.1 ± 1.5 U/mg) was observed.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Generation of Chinese cabbage resistant to bacterial soft rot by heterologous expression of <Emphasis Type="Italic">Arabidopsis WRKY75</Emphasis>

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is a serious disease in Chinese cabbage (Brassica rapa L. subsp. pekinensis). To reduce the severity of soft rot symptoms in Chinese cabbage, Arabidopsis AtWRKY75 was introduced into Chinese cabbage by Agrobacterium-mediated transformation, which was previously reported to reduce susceptibility to Pcc infection in Arabidopsis. Three independent Chinese cabbage transgenic lines carrying AtWRKY75 were obtained. The growth phenotypes of AtWRKY75 overexpression (OE) lines were normal. Bacterial soft rot symptoms and Pcc growth were reduced in AtWRKY75-OE Chinese cabbage lines compared with WT plants. In contrast, overexpression of AtWRKY75 had no effect on infection with a hemibiotrophic pathogen, Xanthomonas campestris pv. campestris (Xcc) causing black rot disease. These results are consistent with those observed in the transgenic Arabidopsis. We found that AtWRKY75 activated a subset of Chinese cabbage genes related to defense against Pcc infection, such as Meri15B, BrPR4, and BrPDF1.2 (but not BrPGIP2). Moreover, overexpression of AtWRKY75 caused H₂O₂ production and activation of H₂O₂ scavenge enzyme genes, suggesting that H₂O₂ played a role in AtWRKY75-mediated resistance to Pcc. Together, these results demonstrated that AtWRKY75 decreased the severity of Pcc-caused bacterial soft rot and activated a subset of Pcc infection defense-related genes in Chinese cabbage similar to in Arabidopsis. It is suggested that AtWRKY75 is a candidate gene for use in crop improvement, because it results in reduced severity of disease symptoms without concurrent growth abnormalities.     

Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells Capable and Incapable of Phenylalanine Production

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the _aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ _aro CP_tac-ARO2 and TG1Δ _aro CP_tac- _aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ _aro CP_tac- _aro C was higher than the level of ARO2 synthesis in the TG1Δ _aro CP_tac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- _aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ _aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ _aro C, which lacked chorismate synthase activity. The similar positive effects of P_tac- _aro C and P_tac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.     

Dietary Iodine Affected the GSH-Px to Regulate the Thyroid Hormones in Thyroid Gland of Rex Rabbits

Iodine (I) is an essential trace element that can influence animal health and productivity. In this study, we investigated the effects of dietary iodine on the antioxidant indices of organ (liver and thyroid gland) and messenger RNA (mRNA) expression of glutathione peroxidase (GSH-Px) in Rex rabbits. A total of 120 4-month-old Rex rabbits (2235.4 ± 13.04 g BW) were divided into four equal groups, and their diets were supplemented with iodine (0, 0.2, 2, or 4 mg/kg dry matter (DM)). The iodine concentration in basal diet (control group) was 0.36 mg/kg DM. In most of measured parameters, supplemental iodine exerted no significant effect. Growth and slaughter performance and organ weight were not influenced significantly by iodine supplementation. Serum T₃ was significantly lower in 2-mg I group than in 0.2 and 4-mg I groups (P < 0.05). Superoxide dismutase (SOD), GSH-Px, methane dicarboxylic aldehyde (MDA), and thyroperoxidase (TPO) in the serum and liver were not influenced (P > 0.05). Conversely, serum catalase (CAT) was significantly reduced (P < 0.05). In the thyroid, GSH-Px was higher in the 2-mg I group than in the 0.2- and 4-mg I groups (P < 0.05). RT-PCR results showed that the mRNA expression level of GSH-Px in the liver was not significantly influenced (P > 0.05). In the thyroid gland, the mRNA expression level of GSH-Px was higher in the 2-mg I group than in the 4-mg I group (P < 0.05), which agreed with the activity of GSH-Px. In conclusion, iodine supplementation exerted no effect on the performance and antioxidant capacity of the body, but dietary iodine influenced serum T₃ or GSH-Px in the thyroid gland. Thus, on the basis of serum T₃ and GSH-Px levels in the thyroid gland, we hypothesized that GSH-Px secretion was increased by adding dietary iodine in the thyroid, which may inhibit the H₂O₂ generation and further influence the thyroid hormone synthesis.     

Thermostability enhancement of chitosanase CsnA by fusion a family 5 carbohydrate-binding module

Objective

To determine the effects of carbohydrate-binding modules (CBMs) on the thermostability and catalytic efficiency of chitosanase CsnA.

Results

Three CBMs (BgCBM5, PfCBM32-2 and AoCBM35) were engineered at the C-terminus of chitosanase CsnA to create hybrid enzymes CsnA-CBM5, CsnA-CBM32 and CsnA-CBM35. K _m values of all the hybrid enzymes were lower than that of the wild type (WT) enzyme; however, only CsnA-CBM5 had an elevated specific activity and catalytic efficiency. The fusion of BgCBM5 enhanced the thermostability of the enzyme, which exhibited a 8.9 °C higher T₅₀ and a 2.9 °C higher T_m than the WT. Secondary structural analysis indicated that appending BgCBM5 at the C-terminus considerably changed the secondary structure content.

Conclusions

The fusion of BgCBM5 improved the thermal stability of CsnA, and the obtained hybrid enzyme (CsnA-CBM5) is a useful candidate for industrial application.

     

Simple generalisation of a mesophyll resistance model for various intracellular arrangements of chloroplasts and mitochondria in C<Subscript>3</Subscript> leaves

The classical definition of mesophyll conductance (g _m) represents an apparent parameter (g _m,app) as it places (photo)respired CO₂ at the same compartment where the carboxylation by Rubisco takes place. Recently, Tholen and co-workers developed a framework, in which g _m better describes a physical diffusional parameter (g _m,dif). They partitioned mesophyll resistance (r _m,dif = 1/g _m,dif) into two components, cell wall and plasmalemma resistance (r _wp) and chloroplast resistance (r _ch), and showed that g _m,app is sensitive to the ratio of photorespiratory (F) and respiratory (R _d) CO₂ release to net CO₂ uptake (A): g _m,app = g _m,dif/[1?+?ω(F?+?R _d)/A], where ω is the fraction of r _ch in r _m,dif. We herein extend the framework further by considering various scenarios for the intracellular arrangement of chloroplasts and mitochondria. We show that the formula of Tholen et al. implies either that mitochondria, where (photo)respired CO₂ is released, locate between the plasmalemma and the chloroplast continuum or that CO₂ in the cytosol is completely mixed. However, the model of Tholen et al. is still valid if ω is replaced by ω(1?σ), where σ is the fraction of (photo)respired CO₂ that experiences r _ch (in addition to r _wp and stomatal resistance) if this CO₂ is to escape from being refixed. Therefore, responses of g _m,app to (F?+?R _d)/A lie somewhere between no sensitivity in the classical method (σ =1) and high sensitivity in the model of Tholen et al. (σ =0).     

Identification of a New 1,4-beta-D-xylosidase <Emphasis Type="Italic">Pae</Emphasis>1263 from the Whole Genome Sequence of <Emphasis Type="Italic">Paenibacillus terrae</Emphasis> HPL-003

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a K _m and V _max of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively.     

Co-expression of Beta-Glucosidase and Laccase in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> by Random Insertion with Enhanced Filter Paper Activity

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P _pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P _gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.     

Fine-scale differences in genetic and census population size ratios between two stream fishes

Comparing the ratio of effective number of breeders (N _b ) to adult population size (N) among closely related coexisting species can provide insights into the role of life history on N _b /N ratios and inform conservation programs towards limiting the loss of evolutionary potential in natural populations. We estimated N _b and N in two coexisting salmonid fishes (Brook trout and Atlantic salmon) for 3–4 consecutive years in two small, adjacent streams in Newfoundland, Canada, using mark-recapture (N), linkage disequilibrium (N _b(LD)), and sibship frequency approaches (N _b(Sib) ). We found that N _b /N ratios were about 20-fold greater in Atlantic salmon than in brook trout (mean 0.20, range 0.06–0.56 vs. mean 0.02, range 0.01–0.05, respectively). This difference was consistent across N _b estimators. In addition, we found that removing migrants reduced N _b : the strength of the effect was weak for N _b(LD) and much stronger for N _b(Sib). Our results highlight the importance of subtle ecological differences and gene flow in shaping N _b /N. They also provide some evidence that the linkage between demographic and evolutionary processes varies between closely related taxa and suggest that a more complete understanding of the N _b /N range across various species is an important component of conservation genetics and management.     

Investigation of the photosynthetic activity of bark phelloderm of arboreous plants using the fluorescent method

Seasonal changes in the characteristics of chlorophyll fluorescence were studied in the bark of several species of trees originating in various climatic zones: Siberian cedar (Pinus sibirica), larch (Larix sibirica), eastern arborvitae (Thuja occidentalis), pendent white birch (Betula pendula), wild black cherry (Padus virginiana), horse chestnut (Aesculus hippocastanum), red oak (Quercus rubra), Manchurian catalpa (Catalpa bungei), linden (Tilia cordata), goat willow (Salix caprea), Amur cherry (Padus maackii), and apple Korichnaya (Malus domestrica B.). Tree bark has a sufficient amount of chlorophyll for measuring the parameters of chlorophyll fluorescence throughout the year. The relative yield of the variable fluorescence of chlorophyll (F _v/F _m) can be used to assess seasonal changes in the physiological state of various trees.     

Another look at the interaction between mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> and flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>

Yeast flavocytochrome b ₂ tranfers reducing equivalents from lactate to oxygen via cytochrome c and cytochrome c oxidase. The enzyme catalytic cycle includes FMN reduction by lactate and reoxidation by intramolecular electron transfer to heme b ₂. Each subunit of the soluble tetrameric enzyme consists of an N terminal b ₅-like heme-binding domain and a C terminal flavodehydrogenase. In the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. This raises a central question: is this mobility required for interaction with the physiological acceptor cytochrome c, which only receives electrons from the heme and not from the FMN? The present review summarizes the results of the variety of methods used over the years that shed light on the interactions between the flavin and heme domains and between the enzyme and cytochrome c. The conclusion is that one should consider the interaction between the flavin and heme domains as a transient one, and that the cytochrome c and the flavin domain docking areas on the heme b ₂ domain must overlap at least in part. The heme domain mobility is an essential component of the flavocytochrome b ₂ functioning. In this respect, the enzyme bears similarity to a variety of redox enzyme systems, in particular those in which a cytochrome b ₅-like domain is fused to proteins carrying other redox functions.     

Molecular cloning and characterization of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase gene in rubber tree (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis>)

S-Adenosylmethionine decarboxylase (SAMDC) is a key rate-limiting enzyme involved in polyamines biosynthesis, and it plays important roles in plant growth, development and stresses response. However, no SAMDC gene was reported in rubber tree. Here we report characteristics of an SAMDC gene (HbSAMDC1) in rubber tree. HbSAMDC1 contains a 1080 bp open reading frame (ORF) encoding 359 amino acids. Quantitative real-time PCR analyses revealed that HbSAMDC1 exhibited distinct expression patterns in different tissues and was regulated by various stresses, including drought, cold, salt, wounding, and H₂O₂ treatments. HbSAMDC1 5′ untranslated region (UTR) contains a highly conserved overlapping tiny and small upstream ORFs (uORFs), encoding 2 and 52 amino acid residues, respectively. No introns were located in the main ORF of HbSAMDC1, whereas two introns were found in the 5′ UTR. In transgenic tobaccos, the highly conserved small uORF of HbSAMDC1 is found to be responsible for translational repression of downstream β-glucuronidase reporter. To our knowledge, this is the first report on molecular cloning, expression profiles, and 5′ UTR characteristics of HbSAMDC1. These results lay solid foundation for further elucidating HbSAMDC1 function in rubber tree.     

Overexpression of the receptor-like cytoplasmic kinase gene <Emphasis Type="Italic">XCRK</Emphasis> enhances Xoc and oxidative stress tolerance in rice

In this paper, we characterized a differentially expressed receptor-like cytoplasmic kinase XCRK, which confers resistance to bacterial leaf streak (BLS). We analyzed the tissue expression of XCRK and showed that XCRK was widely expressed in multiple rice (Oryza sativa) organs, including internodes, roots, leaves and flowers. In addition, the expression of XCRK was significantly induced by ABA, salt and H₂O₂ treatments, suggesting its function in these pathways. The XCRK-overexpressing transgenic seedlings exhibited higher tolerance to Xanthomonas oryzae pv.oryzicola (Xoc) compared with the wild-type seedlings. Furthermore, XCRK-overexpressing seedlings showed stronger antioxidant capacity with reduced MDA and H₂O₂ content and higher antioxidant enzyme activities. It has been hypothesized that the enhanced Xoc tolerance was attributed to the improved expression of resistance-responsive factors positively regulated by XCRK. In accordance with this, the expression of resistance and oxidation-related genes Wrky77, Wrky13, PAL1, PR5, Fe-SOD and SodCc2 were up-regulated by the overexpression of XCRK, which might contribute collectively to the increased Xoc tolerance. Overall, overexpression of XCRK could enhance the antioxidant capacity and Xoc tolerance in rice.     

Kinetic characterization of a novel acid ectophosphatase from <Emphasis Type="Italic">Enterobacter asburiae</Emphasis>

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10⁸ cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows “Michaelis-Menten” kinetics with V = 61.2 U/mg and K_0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K_0.5 = 110 μM, and n_H = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K_0.5 = 84 μM, and n_H = 2.3. Arsenate and phosphate were competitive inhibitors with Ki = 0.6 mM and K_i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K_0.5 = 2.2 mM and n_H = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.     

Anomalous pinch in the T-11M tokamak in an enhanced-collisionality regime

The formation of a peaked bell-shaped profile of the electron density n _e (r) in the T-11M tokamak (B _t=1 T, R/a = 0.7/0.2 m, I _p = 100 kA, t _shot ≤ 300 ms, Li and C limiters) was observed in Li experiments carried out in the near-plateau collisionality regime (the collisionality parameter at one-half of the minor radius was v* ≥ 0.5) under the conditions of low hydrogen recycling and intense hydrogen influx from the plasma edge. It is well known that peaked n _e (r) profiles are observed in collisionless regimes at v* values as low as 10^?1–10^?2 or in impurity-contaminated discharges, in which this effect can be attributed to the impurity accumulation on the plasma column axis. Moreover, a bell-shaped n _e (r) profile in discharges with low n _e can result from the ionization of hydrogen atoms at the column axis, where they arrive from the plasma edge due to cascade charge-exchange. In quasi-steady lithium discharges in T-11M, however, peaked n _e (r) profiles were observed at a relatively high central electron density n _e (0) and relatively high collision frequency, such that the influence of impurities on the n _e (r) profile could be ignored (Z _eff = 1.1±0.1). To explain this effect, one has to assume that the pinching of hydrogen ions in T-11M is anomalous. The lower estimate of the observed pinch velocity is 4 ± 1 m/s, which is three to five times higher than the velocity of the neoclassical (Ware) pinch, characteristic of these conditions. The work is devoted to the experimental study of this effect.