Fluctuation in amino acids content in Triticum aestivum L. cultivars as an indicator on the impact of post-emergence herbicides in controlling weeds

This study was carried out in a demonstrated field in El-Sharkia Governorate, Egypt, during the winter of season 2020 to evaluate the leverage of four post-emergence herbicides i.e., tribenuron-methyl, clodinafop- propargyl, pyroxsulam and pinoxaden compared to control on total protein and amino acid contents in three wheat cultivars (Shandwel 1, Giza 171, and Sakha 95). Generally, the use of foliar herbicides led to a significant decrease in essential, non-essential amino acids and protein contents. However, tribenuran-methyl herbicide signifcantly increased the levels of proline, glycine, arginine, and histidine, but cystine and threonine not affected as compared to control. On the other hand, foliar herbicide application was significantly increased physiological , biochemical parameters and yield of Shandweel cultivar as compared to the other varieties. The physiological and biochemical models of dual-herbicide-tolerant wheat cultivars add to our understanding of the crop. In recent agricultural systems, herbicide tolerant plants are important for long-term weed management. Therefore, the study recommended the safely usage of Tribenuran-methyl as foliar herbicide in weed managment.     

Effect of sulfite on compositional changes of cell constituents in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L., which were preincubated at 25 C for three days in the dark, were suspended in 1 mM potassium phosphate buffer, pH 6.0, and incubated for four days under continuous red light in the presence or absence of 3 mM sulfite. At day 0, 2 and 4 of the incubation, contents of cell constituents were determined. Total lipid content decreased continuously over four days of incubation in the absence of sulfite or in the presence of 3 mM sulfate. In contrast, when sulfite was added to the medium, the decrease stopped after day 2. The content of insoluble glucan increased markedly between day 2 and 4 in the medium without sulfite, whereas it decreased continuously for four days in the medium containing sulfite. The protein content decreased promptly by day 2, but its decrease was delayed when 3 mM sulfite was added to the medium. The content of amino acids also decreased by day 2, but it increased thereafter in the absence of sulfite or in the presence of 3 mM sulfate. In the presence of sulfite, however, the content continued to decrease until day 4. The results indicate that 3 mM sulfite in the incubation medium depressed the utilization of reserve lipid and protein, the synthesis of insoluble glucan and the increase of amino acid pool sizes in fern spores.     

Allocation of proteolytic activity in the seedling of Cynara cardunculus L. in the initial growth stages

Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were enhanced, as were contents of all aa except Pro, exhibiting the highest levels in cotyledons on the 15th post-germinative day.     

Stream channel experiments on downstream movement of recently emerged trout, Salmo trutta L. and salmon, S. salar L.—I. Effect of four different water velocity treatments upon dispersal rate

Four experimental stream channels were used to study instantaneous downstream dispersal rates of young trout, Salmo trutta L., and salmon, S. salur L ., relative to four different water velocities.
Young salmon showed a high rate of dispersal at a low velocity of 7.5 cm s⁻¹ and lower rates at higher velocities of 25 to 70cm s⁻¹. Trout showed their lowest rate at 25cm s⁻¹ with a slightly higher rate at 7.5 cm s⁻¹ and increasingly higher rates at velocities in excess of 25 cm s⁻¹. These results are consistent with field observations on the velocity preferences of young trout and salmon.     

Phosphorus deficiency induced by nitrogen input in Douglas fir in the Netherlands

Summary A re-examination of earlier NPK fertilization experiments in Douglas fir stands on sandy soils shows the effects of high nitrogen input by air pollution during the last 10–15 years on plant nutrition at these sites. In 1960, experimental plots showed a positive growth reaction to nitrogen, phosphorus, and potassium fertilization. All suffered from severe phosphorus deficiency in 1984, low phosphorus in the needles was invariably accompanied by a high nitrogen content, with all N/P ratios between 20 and 30. The same conclusion emerges from an independent investigation of nutrient status of a selection of Douglas fir stands. Hence, if stand productivity and a balanced nutrient status of the trees is to be maintained, the increase in atmospheric input of nitrogen calls for supplementary fertilization. Given the current N/P ratios in the needles, a positive growth response to phosphorus fertilization is to be expected.     

营养盐初始组合浓度对类波氏真眼点藻生长和油脂积累的影响

为了研究培养基中主要营养元素氮、磷、硫初始组合浓度对类波氏真眼点藻(Eustigmatos cf.polyphem D.J.Hibberd)生长、油脂积累和脂肪酸组成的影响,分别以NaNO_3、K_2HPO_4、MgSO_4为氮、磷、硫源,设置不同的营养盐初始组合浓度对其进行培养,采用干重法、重量法、气相色谱分析法、元素分析仪等依次对生物量、总脂、脂肪酸组成和细胞内元素含量进行测定。结果显示,氮、磷、硫三者的初始组合浓度变化对类波氏真眼点藻的生长和油脂积累具有明显的影响,9 mmol/L NaNO_3、0.92 mmol/L K_2HPO_4、0.3 mmol/L MgSO_4营养条件下其生物量达到最大值,为9.19 g/L;低氮、低磷胁迫或二者共同胁迫均促进其油脂积累,但低硫胁迫对油脂的积累影响不大,最大油脂含量出现在3 mmol/L NaNO_3、0.029 mmol/L K_2HPO_4、0.3 mmol/L MgSO_4条件下,为68.7%(DW)。此外,类波氏真眼点藻富含棕榈油酸,占总脂肪酸含量的最大值达60.88%。选择合理的氮、磷、硫初始组合浓度能够有效促进其生长或油脂及棕榈油酸的积累。     

The Influence of Nitrogen Nutrition on the Carbohydrate and Nitrogen Status of Emergent Macrophyte Acorus calamus L.

Carbon and nitrogen balance in Acorus calamus, a wetland species colonising littoral zones with a high trophic status, was studied under experimental conditions using water or sand culture with a defined composition of the nutrient solution. Influence of graded level of N (1.86, 7.5 and 18.6 mM) and/or forms of N (NH₄⁺ versus NO ₃^–) on the content of non-structural carbohydrates, free amino acids, total C, and total N was studied in Acorus rhizomes and roots to find possible connection with a reduced growth of Acorus plants under high N and NH₄⁺–N nutrition described in our previous study [Vojtíšková et al., 2004. Hydrobiologia 518: 9–22]. High N availability and pure NH₄⁺–N nutrition affected the C/N balance of rhizome and root systems of Acorus in a similar way. NH₄⁺–N was the only form of N elevated under the high N treatment. The major proportion of the total non-structural carbohydrates (TNC) was starch (91–93% and 51–64% in rhizomes and roots, respectively). The content of starch was significantly and and negatively affected by high N availability (P = 0.001), as well as by NH₄⁺–N nutrition (P=0.001). Amounts of simple soluble carbohydrates (sucrose, glucose, and fructose) were negligible in comparison to starch in rhizomes and branched roots (up to 5% of TNC), while roots without developed lateral roots (unbranched) contained up to 33% of TNC in the form of simple soluble sugars. Moreover, high hexoses/sucrose ratio, low starch/soluble sugars ratio, high content of N, and low C/N ratio support the notion that unbranched roots are metabolically active young roots with tissue differentiation in progress. A high content of free amino acids, typically with dominance of N-rich amino acids (Arg-46%, Gln-8%, Asn-7%), was found simultaneously with a low carbohydrate content under high N supply, which indicates that NH₄⁺ received is effectively incorporated into the organic form by this species. Since the decrease in carbohydrate content was not accompanied by luxurious growth, other possible carbon consuming processes were discussed in relation to NH₄⁺ nutrition. More dramatic changes in total N than C were found under high N availability resulting a shift in C/N ratio in favour of N. Although the shift towards N metabolism was obvious, no serious carbohydrate depletion occurred, which could explain the reduced growth of Acorus plants under high N and sole NH₄⁺–N nutrition described previously.     

Effect of C/N ratio and aeration on the fatty acid composition of heterotrophicChlorella sorokiniana

The effect of the carbon to nitrogen (C/N) ratio of the medium and the aeration rate on the lipid content and fatty acid composition ofChlorella sorokiniana was investigated using heterotrophic, batch culture. Both parameters had a significant effect. A C/N ratio of approximately 20, was found to indicate a change from carbon to nitrogen limitation forC. sorokiniana. Cell lipid content was at a minimum at this value and increased at both higher and lower C/N values. Low C/N ratios favoured a high proportion of trienoic fatty acids at the expense of monoenoic acids. Aeration enhanced cell growth, fatty acid yield and the synthesis of unsaturated dienoic and trienoic fatty acids, but reduced cell lipid content. The results demonstrate that the fatty acid composition and lipid content of heterotrophically-grown microalgae can be favourably manipulated by varying culture conditions.     

Variation in tissue element concentrations in Quercus ilex L. over a range of different soils

In order to study the variability in nutrient concentrations in four tissues of Q. ilex in relation to soil properties, we selected fifteen stands in both Quercus ilex forests and Q. ilex-Pinus halepensis mixed forests. These stands had developed on soils derived from eight different parent materials. Three soil groups were differentiated according to their chemical properties: calcareous soils, siliceous soils, and volcanic soils. Across sites, nutrient concentrations were generally less variable in current-year tissues than in older tissues. Nitrogen and potassium showed the lowest variability among sites, their concentrations in current-year leaves ranging from 1.17% to 1.39% for N and from 0.53% to 0.68% for K. There were few statistically significant correlations between tissue element concentrations, the most frequent being the antagonistic relationship between calcium and magnesium. Nitrogen concentration in current-year leaves was negatively correlated with soil chemical fertility (nitrogen, phosphorus and potassium). This may reflect a nutritional imbalance between nitrogen and other nutrients, some of which may be more limiting than nitrogen to Q. ilex growth in Catalonia forests. Negative correlations were also found between plant magnesium and soil calcium, and positive correlations between plant calcium and soil calcium.     

The effect of elevated CO2 on the chemical composition and construction costs of leaves of 27 C3 species

We determined the proximate chemical composition as well as the construction costs of leaves of 27 species, grown at ambient and at a twice-ambient partial pressure of atmospheric CO₂. These species comprised wild and agricultural herbaceous plants as well as tree seedlings. Both average responses across species and the range in response were considered. Expressed on a total dry weight basis, the main change in chemical composition due to CO₂ was the accumulation of total non-structural carbohydrates (TNC). To a lesser extent, decreases were found for organic N compounds and minerals. Hardly any change was observed for total structural carbohydrates (cellulose plus hemicellulose), lignin and lipids. When expressed on a TNC-free basis, decreases in organic N compounds and minerals were still present. On this basis, there was also an increase in the concentration of soluble phenolics. In terms of glucose required for biosynthesis, the increase in costs for one chemical compound – TNC – was balanced by a decrease in the costs for organic N compounds. Therefore, the construction costs, the total amount of glucose required to produce 1 g of leaf, were rather similar for the two CO₂ treatments; on average a small decrease of 3% was found. This decrease was attributable to a decrease of up to 30% in the growth respiration coefficient, the total CO₂ respired [mainly for N AD(P)H and ATP] in the process of constructing 1 g of biomass. The main reasons for this reduction were the decrease in organic N compounds and the increase in TNC.     

Effect of varying Mg/Ca ratio and electrolyte concentration in the irrigation water on the soil properties and growth of wheat

This paper discusses the results of a pot experiment conducted to study the effect of irrigation waters having varying Mg/Ca ratio (2, 4, 8 and 16) and electrolyte concentration (20 and 80 meq/l) on the soil properties and growth of wheat crop in two different soils. The development of salinity in the soils generally increased at higher electrolyte concentration of the irrigation water, but it was of a greater magnitude in the heavy-textured black soil dominated by montmorillonite clay mineral than in the light-textured alluvial soil having illite type of clay mineral. The accumulation of soluble salts as a result of saline water irrigation was higher in the surface layer than in the subsurface layer in both soils. The adsorption of Na and Mg in the soils increased with an increase in the Mg/Ca ratio and electrolyte concentration of the irrigation water. These changes in soil properties were adequately reflected by the grain and dry matter yields of wheat crop, which showed a significant reduction with an increase in the Mg/Ca ratio and electrolyte concentration of the irrigation water. However, the effects of these treatments were more pronounced in the heavy black clay soil than in the alluvial soil. Thus, the role of Mg is different from that of Ca under the conditions used in the experiment.     

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Nitrate reduction in roots and shoots and exchange of reduced N between organs were quantitatively estimated in intact 13-d-old seedlings of two-row barley (Hordeum vulgare L. cv. Daisengold) using the ¹⁵N-incorporation model (A. Gojon et al. (1986) Plant Physiol. 82, 254–260), except that NH ₊ ⁴ was replaced by NO _- ² . N-depleted seedlings were exposed to media containing both nitrate (1.8 mM) and nitrite (0.2 mM) under a light-dark cycle of 12:12 h at 20°C; the media contained different amounts of ¹⁵N labeling. Experiments were started either immediately after the beginning (expt. 1) or immediately prior to the end (expt. 2) of the light period, and plants were sampled subsequently at each light-dark transition throughout 36 h. The plants effectively utilized ¹⁵NO _- ³ and accumulated it as reduced ¹⁵N, predominantly in the shoots. Accumulation of reduced ¹⁵N in both experiments was nearly the same at the end of the experiment but the accumulation pattern in roots and shoots during each 12-h period differed greatly depending on time and the light conditions. In expt. 1, the roots accounted for 31% (light), 58% (dark), and 9% (light) of nitrate reduction by the whole plants, while in expt. 2 the contributions of the root were 82% (dark), 20% (light), and 29% (dark), during each of the three 12-h periods. Xylem transport of nitrate drastically decreased in the dark, but that of reduced N rather increased. The downward translocation of reduced ¹⁵N increased while nitrate reduction in the root decreased, whereas upward translocation decreased while nitrate reduction in the shoot increased. We conclude that the cycling of reduced N through the plant is important for N feeding of each organ, and that the transport system of reduced N by way of xylem and phloem, as well as nitrate reduction by root and shoot, can be modulated in response to the relative magnitude of reduced-N demands by the root and shoot, with the one or the other predominating under different circumstances.Symbols Anl accumulation of reduced ¹⁵N from ¹⁵NO _- ³ in ¹⁴NO _- ³ -fed roots of divided root system - Ar accumulation in root of reduced ¹⁵N from ¹⁵NO _- ³ - As accumulation in shoot of reduced ¹⁵N from ¹⁵NO _- ³ - Rr ¹⁵NO _- ³ reduction in root - Rs ¹⁵NO _- ³ reduction in shoot - Tp translocation to root of shoot-reduced ¹⁵N from ¹⁵NO _- ³ in phloem - Tx translocation to shoot of root-reduced ¹⁵N from ¹⁵NO _- ³ in xylem     

Effects of maturity stage, wilting and acid treatment on crude protein fractions and chemical composition of whole crop pea silages (Pisum sativum L.)

In order to determine if maturity stage, and wilting or acid treatment, change the crude protein (CP) fraction distribution (determined according to the Cornell Net Carbohydrate and Protein System) of whole crop pea silages, a pea with variegated flowers (Pisum sativum ssp. arvense L., cv Timo) was compared to a white-flowered, semi-leafless pea (P. sativum ssp. hortense L, cv Capella). Herbage was harvested at three maturity stages being: pod set, pod swell and full pod, and either acid-treated or wilted. Timo was acid-treated using 4 (acid4), 6 (acid6) or 8 (acid8) L/tonne fresh matter (FM) with a 2:1 mixture of formic and propionic acid, or wilted to a dry matter (DM) content of about 400 g/kg. Capella was treated with acid6 or wilted. Herbage was ensiled for 103 days in 10 kg laboratory silos. Despite differences in wilting conditions, all wilted herbages had similar protein fraction distributions. In the Capella silages the soluble CP content was lower in the later maturity stages, but this was not the case in the Timo silages. The amount of acid added only affected the B1 CP fraction content, which decreased with increasing acid. At pod set and pod swell for Timo, and at pod set for Capella, the direct-harvested herbages were difficult to ensile because of the high buffering capacity and low level of water soluble carbohydrates. Wilting improved ensilability. Acid treatment reduced proteolysis, but crops with DM contents below 150 g/kg must be acid treated with at least 6 L/tonne FM to ensure stable fermentation. Timo silages were more prone to malfermentation, probably caused by lodging, which made Capella the preferred cultivar for producing pea silages harvested at the pod swell stage or later. Proteolysis and the amount of soluble CP in the silage were lower in later maturity stages in the Capella, but not the Timo, cultivar.     

Amide proton exchange measurements as a probe of the stability and dynamics of the N-terminal domain of the ribosomal protein L9: comparison with the intact protein.

Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1-56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N-terminal domain are very similar to those previously measured for the corresponding region in the full-length protein (Lillemoen J et al., 1997, J Mol Biol 268:482-493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N-terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.     

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et␣al. (1995, Biochem J 311:805–813) and LaFayette et␣al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions. Received: 11 July 1996 / Accepted: 24 August 1996     

Anaerobic treatment of complex chemical wastewater in a sequencing batch biofilm reactor: process optimization and evaluation of factor interactions using the Taguchi dynamic DOE methodology

The Taguchi robust experimental design (DOE) methodology has been applied on a dynamic anaerobic process treating complex wastewater by an anaerobic sequencing batch biofilm reactor (AnSBBR). For optimizing the process as well as to evaluate the influence of different factors on the process, the uncontrollable (noise) factors have been considered. The Taguchi methodology adopting dynamic approach is the first of its kind for studying anaerobic process evaluation and process optimization. The designed experimental methodology consisted of four phases--planning, conducting, analysis, and validation connected sequence-wise to achieve the overall optimization. In the experimental design, five controllable factors, i.e., organic loading rate (OLR), inlet pH, biodegradability (BOD/COD ratio), temperature, and sulfate concentration, along with the two uncontrollable (noise) factors, volatile fatty acids (VFA) and alkalinity at two levels were considered for optimization of the anae robic system. Thirty-two anaerobic experiments were conducted with a different combination of factors and the results obtained in terms of substrate degradation rates were processed in Qualitek-4 software to study the main effect of individual factors, interaction between the individual factors, and signal-to-noise (S/N) ratio analysis. Attempts were also made to achieve optimum conditions. Studies on the influence of individual factors on process performance revealed the intensive effect of OLR. In multiple factor interaction studies, biodegradability with other factors, such as temperature, pH, and sulfate have shown maximum influence over the process performance. The optimum conditions for the efficient performance of the anaerobic system in treating complex wastewater by considering dynamic (noise) factors obtained are higher organic loading rate of 3.5 Kg COD/m3 day, neutral pH with high biodegradability (BOD/COD ratio of 0.5), along with mesophilic temperature range (40 degrees C), and low sulfate concentration (700 mg/L). The optimization resulted in enhanced anaerobic performance (56.7%) from a substrate degradation rate (SDR) of 1.99 to 3.13 Kg COD/m3 day. Considering the obtained optimum factors, further validation experiments were carried out, which showed enhanced process performance (3.04 Kg COD/m3-day from 1.99 Kg COD/m3 day) accounting for 52.13% improvement with the optimized process conditions. The proposed method facilitated a systematic mathematical approach to understand the complex multi-species manifested anaerobic process treating complex chemical wastewater by considering the uncontrollable factors.     

Plasticity in R/S ratio, morphology and fitness-related traits in response to reciprocal patchiness of light and nutrients in the stoloniferous herb, Glechoma longituba L

Clonal fragments of the stoloniferous herb Glechoma longituba were subjected to a complementary patchiness of light and soil nutrients including two spatially homogeneous treatments (SR–SR and IP–IP) and two spatially heterogeneous treatments (IP–SR and SR–IP). SR and IP indicate patches (shaded, rich) with low light intensity (shaded, S), high nutrient availability (rich, R) and patches (illuminated, poor) with high light intensity (illuminated, I) and low nutrient availability (poor, P), respectively. Plasticity of the species in root–shoot ratio, fitness-related traits (biomass, number of ramets and dry weight per ramet) and clonal morphological traits (length and specific length of stolon internodes, area and specific area of laminae, length and specific length of petioles) were experimentally examined. The aim is to understand adaptation of G. longituba to the environment with reciprocal patches of light and soil nutrients by plasticities both in root–shoot ratio and in (clonal) morphology. Our experiment revealed performance of the clonal fragments growing from patches with high light intensity and low soil nutrient availability into the adjacent opposite patches was increased in terms of the fitness-related characters. R/S ratio and clonal morphology were plastic. Meanwhile, the capture of light resource from the light-rich patches was enhanced while the capture of soil nutrients from either the nutrient-rich or the nutrient-poor patches was not. Analysis of cost and benefit disclosed positive effects of clonal integration on biomass production of ramets in the patches with low light intensity and high soil nutrient availability. These results suggest an existence of reciprocal translocation of assimilates and nutrients between the interconnected ramets. The reinforced performance of the clonal fragments seems to be related with specialization of clonal morphology in the species.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability

Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of alpha-1,6-branch points. They are the members of the alpha-amylase family and have four conserved regions in a central (beta/alpha)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (DeltaN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same K(m) values as PvSBE2 but had lower V(max) values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.