Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

Spawning habits and embryonic development of the banded lampeye killifish Aplocheilichthys spilauchen (Duméril 1861) in ex situ fresh and brackish water environments

Aside from ornamental uses, there is growing interest in using killifishes for a multiplicity of purposes including baitfish and mosquito biocontrol. This experiment explored the spawning habits and embryonic development of the banded lampeye, Aplocheilichthys spilauchen in ex situ freshwater (0.04‰) and brackish water (5.01‰) to ascertain the captive breeding prospects for mosquito control in areas where they occur. Significantly higher number of eggs were laid in the brackish water than the freshwater (X² = 1613.0, P < 0.05), and black mop was the most preferred spawning substrate, followed by green, blue and white mops. Microscopic monitoring of embryos revealed that cleavage occurred within the first 30 min after fertilization, organogenesis commenced on average in the 25th hour and hatching in approximately 230 h. Although freshwater eggs were relatively bigger than brackish water eggs and certain embryonic developmental stages occurred faster in the freshwater than brackish water, these differences were overall not significant and had no effects on the development and hatching. The observed outcome that A. spilauchen can be optimally propagated with black mops in brackish water offers a significant step in its use for the mosquito biocontrol programme, as well as other potential uses not yet explored.     

Optimal Design of a Series of GSTR's Performing Reversible Reactions Catalyzed by Soluble Enzymes: A Theoretical Study

The condition for the minimum overall reactor volume of a given number of CSTR's in series is theoretically determined for a reversible, single reactant-single product (Uni-Uni) enzyme catalyzed reaction. The reactor network is assumed to operate in steady-state, isothermal conditions with a single phase and a constant activity of biocatalyst. The method is based on a mathematical analysis of the discrete substrate concentration profile along the CSTR's assuming complete micromixing. The algebraic equations describing the critical loci are obtained for the general case, the mathematical proof that these equations define a minimum is presented, and an exact solution arising from an asymptotic situation is found. An approximate analytical method of optimization based on the aforementioned critical behavior is reported and its validity and usefulness discussed. The formulae introduced can be used in more general situations as tools for getting the approximate range where the optimal overall volume of the series of CSTR's lies. Hence, the reasoning developed is important for the preliminary CSTR design and relevant in the initial steps of the more involved methods of numerical optimization. Finally, the enzymatic conversion of fumarate to L-malate is examined as a model system in order to assess the usefulness and applicability of the analysis developed.     

On the Prediction of the Enantioselectivity of Candida rugosa Lipase by Comparative Molecular Field Analysis

Comparative molecular field analysis (CoMFA) was used to derive a quantitative substrate model for the enzymatic resolution of norbornanol esters by the lipase of Candida rugosa. Within these calculations, the steric and electrostatic interactions of both enantiomers and the differences in the corresponding two fields were used for the correlation of the substrate structure with the enantioselectivity of the hydrolysis. Different alignments of the molecules were used to improve the model.     

Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from Trametes multicolor

Site-directed mutagenesis was used to enhance the catalytic activity of pyranose 2-oxidase (P2Ox) from Trametes multicolor with different substrates. To this end, threonine at position 169 was replaced by glycine, alanine and serine, respectively. Using oxygen as electron acceptor the mutant T169G was equally active with d-glucose and d-galactose, whereas wild-type recombinant P2Ox only showed 5.2% relative activity with the latter substrate. When d-galactose was used as electron donor in saturating concentrations, T169G showed a 4.5-fold increase in its catalytic efficiency k_cat/K_M for the alternative electron acceptor 1,4-benzoquinone and a nine-fold increased k_cat/K_M value with the ferricenium ion compared with wt recP2Ox. Variant T169S showed an increase in its catalytic efficiency both with 1,4-benzoquinone (3.7 times) as well as with the ferricenium ion (1.4 times) when d-glucose was the substrate.     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

Quantifying the inactivation rate constants for the molecular species comprising the catalytic cycle of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

When an unstable enzyme is incubated with its substrate(s), catalysis may cease before chemical equilibrium is attained. The residual substrate concentrations depend on their initial concentrations, the initial enzymic activity, and the inactivation rate constants for each molecular species that comprise the catalytic cycle. The underlying theory has been elaborated previously for single-substrate reactions and here it is extended to bi-substrate reactions. The theory is illustrated by application to glucose 6-phosphate dehydrogenase, which is unstable when exposed to a low concentration of sodium dodecyl sulphate. It is shown that the ternary complex containing both substrates is resistant to inactivation while each of the remaining complexes undergoes first-order decay. Rate constants for the inactivation of each complex are calculated.     

Phytogeographical affinities at the crossroads of two continents: Distribution patterns of Lamiaceae in Chios Island (East Aegean Islands,Greece) and Çeşme–Karaburun Peninsula (West Anatolia,Turkey)

The East Aegean (East Aegean Islands, Greece and West Anatolia, Turkey) is a biogeographically transitional region, where biodiversity elements from Europe and Asia join. However, affinities in the region were until recently scarcely explored. We assess biogeographical affinities in the East Aegean focusing on distribution patterns of Lamiaceae plants in Chios Island and its adjacent Çe?me–Karaburun Peninsula. Detailed in-situ record was acquired for 48 native species. These were grouped based on their habitat and geological substrate preference, their distribution was mapped in grid cells and distribution patterns were analysed in relation to species groupings. In both Chios and Çe?me–Karaburun, species follow five distribution patterns: widespread, locally widespread, locally restricted, sporadic and rare. Fifty to 62% of the species exhibit similar distribution patterns, trends in habitat and geological substrate preference in Chios and Çe?me–Karaburun, results complying with previous evidence of close biogeographical affinities of the East Aegean Islands and neighbouring Anatolia. Differences observed between the two regions may be attributed to insularity effects, human impact and the melange, an old rock matrix known for its key role in elucidating regional geodynamic evolution. Distributions of widespread and locally widespread species in Chios give evidence of density compensation and niche shifts responses, however, the actual occurrence of these phenomena in island plant populations is still to be elucidated. Overall, the species distribution patterns, particularly those of rare ones, reflect the complex geological history, palaeogeography and human influence in the Aegean region.     

Vegetation feedbacks of nutrient addition lead to a weaker carbon sink in an ombrotrophic bog

To study vegetation feedbacks of nutrient addition on carbon sequestration capacity, we investigated vegetation and ecosystem CO₂ exchange at Mer Bleue Bog, Canada in plots that had been fertilized with nitrogen (N) or with N plus phosphorus (P) and potassium (K) for 7–12 years. Gross photosynthesis, ecosystem respiration, and net CO₂ exchange were measured weekly during May–September 2011 using climate‐controlled chambers. A substrate‐induced respiration technique was used to determine the functional ability of the microbial community. The highest N and NPK additions were associated with 40% less net CO₂ uptake than the control. In the NPK additions, a diminished C sink potential was due to a 20–30% increase in ecosystem respiration, while gross photosynthesis rates did not change as greater vascular plant biomass compensated for the decrease in Sphagnum mosses. In the highest N‐only treatment, small reductions in gross photosynthesis and no change in ecosystem respiration led to the reduced C sink. Substrate‐induced microbial respiration was significantly higher in all levels of NPK additions compared with control. The temperature sensitivity of respiration in the plots was lower with increasing cumulative N load, suggesting more labile sources of respired CO₂. The weaker C sink potential could be explained by changes in nutrient availability, higher woody : foliar ratio, moss loss, and enhanced decomposition. Stronger responses to NPK fertilization than to N‐only fertilization for both shrub biomass production and decomposition suggest that the bog ecosystem is N‐P/K colimited rather than N‐limited. Negative effects of further N‐only deposition were indicated by delayed spring CO₂ uptake. In contrast to forests, increased wood formation and surface litter accumulation in bogs seem to reduce the C sink potential owing to the loss of peat‐forming Sphagnum.     

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.