Enzyme Stoichiometry Research Articles

Dominant mycorrhizal association of trees alters carbon and nutrient cycling by selecting for microbial groups with distinct enzyme function.

While it is well established that plants associating with arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi cycle carbon (C) and nutrients in distinct ways, we have a limited understanding of whether varying abundance of ECM and AM plants in a stand can provide integrative proxies for key biogeochemical processes. We explored linkages between the relative abundance of AM and ECM trees and microbial functioning in three hardwood forests in southern Indiana, USA. Across each site's 'mycorrhizal gradient', we measured fungal biomass, fungal:bacterial (F:B) ratios, extracellular enzyme activities, soil carbon:nitrogen ratio, and soil pH over a growing season. We show that the percentage of AM or ECM trees in a plot promotes microbial communities that both reflect and determine the C to nutrient balance in soil. Soils dominated by ECM trees had higher F:B ratios and more standing fungal biomass than AM stands. Enzyme stoichiometry in ECM soils shifted to higher investment in extracellular enzymes needed for nitrogen and phosphorus acquisition than in C-acquisition enzymes, relative to AM soils. Our results suggest that knowledge of mycorrhizal dominance at the stand or landscape scale may provide a unifying framework for linking plant and microbial community dynamics, and predicting their effects on ecological function.

Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design.

The production of natural product compound libraries has been observed in nature for different organisms such as bacteria, fungi and plants; however, little is known about the mechanisms generating such chemically diverse libraries. Here we report mechanisms leading to the biosynthesis of the chemically diverse rhabdopeptide/xenortide peptides (RXPs). They are exclusively present in entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes delivering them to insect prey, which is killed and utilized for nutrition by both nematodes and bacteria. Chemical diversity of the biologically active RXPs results from a combination of iterative and flexible use of monomodular nonribosomal peptide synthetases including substrate promiscuity, enzyme cross-talk and enzyme stoichiometry as shown by in vivo and in vitro experiments. Together, this highlights several of nature's methods for diversification, or evolution, of natural products and sheds light on the biosynthesis of the bioactive RXPs.

Soil enzyme activity and stoichiometry in forest ecosystems along the North-South Transect in eastern China (NSTEC)

Soil enzymes, as indicators of microbial metabolism, play an important role in nutrient biogeochemistry at the ecosystem level. In this study, we present information from a comprehensive analysis of the latitudinal variations in, and stoichiometric relationships between, soil β-glucosidase (BG), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and acid phosphatase (AP) in nine forest ecosystems along the North-South Transect in eastern China. The results showed that soil BG and NAG activities were higher in temperate forests than in subtropical and tropical forests. Soil AP activities were the opposite, which indicates that microbial nutrient demand in tropical forests was limited by the nutrient phosphorus (P). Soil BG and NAG activities were significantly and negatively correlated with mean annual temperature (MAT), mean annual precipitation (MAP), the soil carbon (C):P and soil nitrogen (N):P ratios, but not with the soil C:N ratio. Soil NAG and AP activities were inversely correlated with soil pH, and soil AP activity increased as soil pH decreased. The latitudinal variations in the C:N, C:P, and N:P acquisition ratios represented by ln(BG):ln(LAP + NAG):ln(AP) activities across ecosystems were significantly and negatively related to MAP and MAT. The C:P and N:P acquisition ratios were positively related to soil pH but negatively related to the soil C:P and N:P ratios. The C:N and C:P acquisition ratios were also negatively correlated with leaf C:N, C:P, and N:P ratios. This study provides useful information about environmental controls on enzyme stoichiometry, and also highlights the stoichiometric and energy limitations on the metabolism of soil microbes.

Influence of Macrofaunal Burrows on Extracellular Enzyme Activity and Microbial Abundance in Subtropical Mangrove Sediment.

Bioturbation and bioirrigation induced by burrowing macrofauna are recognized as important processes in aquatic sediment since macrofaunal activities lead to the alteration of sediment characteristics. However, there is a lack of information on how macrofauna influence microbial abundance and extracellular enzyme activity in mangrove sediment. In this study, the environmental parameters, extracellular enzyme activities, and microbial abundance were determined and their relationships were explored. Sediment samples were taken from the surface (S) and lower layer (L) without burrow, as well as crab burrow wall (W) and bottom of crab burrow (B) located at the Mai Po Nature Reserve, Hong Kong. The results showed that the burrowing crabs could enhance the activities of oxidase and hydrolases. The highest activities of phenol oxidase and acid phosphatase were generally observed in B sediment, while the highest activity of N-acetyl-glucosaminidase was found in W sediment. The enzymatic stoichiometry indicated that the crab-affected sediment had similar microbial nitrogen (N) and phosphorous (P) availability relative to carbon (C), lower than S but higher than L sediment. Furthermore, it was found that the highest abundance of both bacteria and fungi was shown in S sediment, and B sediment presented the lowest abundance. Moreover, the concentrations of phosphorus and soluble phenolics in crab-affected sediment were almost higher than the non-affected sediment. The alterations of phenolics, C/P and N/P ratios as well as undetermined environmental factors by the activities of crabs might be the main reasons for the changes of enzyme activity and microbial abundance. Finally, due to the important role of phenol oxidase and hydrolases in sediment organic matter (SOM) decomposition, it is necessary to take macrofaunal activities into consideration when estimating the C budget in mangrove ecosystem in the future.

Nutrient limitation status in a subtropical mangrove ecosystem revealed by analysis of enzymatic stoichiometry and microbial abundance for sediment carbon cycling

Mangrove ecosystem plays an important role in global cycling of nutrients. According to nutrient availability, microorganisms can optimize the allocation of C-, N- and P-degrading enzymes. Thus, in this study, enzymatic stoichiometry involved in C, N and P acquisition, and the microbial abundance were analysed in coastal mangrove sediments in two different seasons. Activities of β-1,4-glucosidase (GLU), N-acetyl-β-glucosaminidase (NAG), acid phosphotase (ACP) and phenol oxidase (PHO) were determined, and then applied to calculate the enzymatic C:N (GLU:NAG), C:P (GLU:ACP) and N:P (NAG:ACP) acquisition, respectively. Besides, GLU:PHO was applied as relative recalcitrance of sediment organic matter (SOM). The results showed the enzymatic stoichiometry and microbial abundance had strongly seasonal variations. Compared to other ecosystems, GLU:ACP and NAG:ACP ratios were largely lower, suggesting that sediments in this ecosystem might be microbial P-limitation. Moreover, the higher GLU:PHO ratio implied that SOM in this ecosystem might be more decomposable. Furthermore, the imbalance of nutrients for microbial abundance was revealed by the strong correlations relating sediment N:P to GLU:NAG, GLU:ACP, and GLU:PHO. On the other hand, microbial P limitation in this ecosystem may enhance C storage and sequestration because the higher C contents in sediments of March than November occurred with lower GLU:ACP and NAG:ACP ratios as well as bacterial abundance.

Model-Based Design of Biochemical Microreactors.

Mathematical modeling of biochemical pathways is an important resource in Synthetic Biology, as the predictive power of simulating synthetic pathways represents an important step in the design of synthetic metabolons. In this paper, we are concerned with the mathematical modeling, simulation, and optimization of metabolic processes in biochemical microreactors able to carry out enzymatic reactions and to exchange metabolites with their surrounding medium. The results of the reported modeling approach are incorporated in the design of the first microreactor prototypes that are under construction. These microreactors consist of compartments separated by membranes carrying specific transporters for the input of substrates and export of products. Inside the compartments of the reactor multienzyme complexes assembled on nano-beads by peptide adapters are used to carry out metabolic reactions. The spatially resolved mathematical model describing the ongoing processes consists of a system of diffusion equations together with boundary and initial conditions. The boundary conditions model the exchange of metabolites with the neighboring compartments and the reactions at the surface of the nano-beads carrying the multienzyme complexes. Efficient and accurate approaches for numerical simulation of the mathematical model and for optimal design of the microreactor are developed. As a proof-of-concept scenario, a synthetic pathway for the conversion of sucrose to glucose-6-phosphate (G6P) was chosen. In this context, the mathematical model is employed to compute the spatio-temporal distributions of the metabolite concentrations, as well as application relevant quantities like the outflow rate of G6P. These computations are performed for different scenarios, where the number of beads as well as their loading capacity are varied. The computed metabolite distributions show spatial patterns, which differ for different experimental arrangements. Furthermore, the total output of G6P increases for scenarios where microcompartimentation of enzymes occurs. These results show that spatially resolved models are needed in the description of the conversion processes. Finally, the enzyme stoichiometry on the nano-beads is determined, which maximizes the production of glucose-6-phosphate.

Differential Distribution of Metals and Enzymes in Quanzhou Bay Estuarine Wetland Soils under Three Mangrove Species

This study aimed to investigate the effects of three dominant mangrove species (Avicennia marina, Aegiceras corniculatum, and Kandelia obovata) on the distribution of the metal elements Fe, Mn, Cu, and Zn and the enzymes urease, phosphatase, polyphenol oxidase, and catalase. Concentrations of the metallic elements and enzymatic activities were quantified in soils under the three mangrove species in the Quanzhou Bay estuarine wetland, a typical coastal wetland in China. Results showed that A. corniculatum promoted aggregation of Mn, urease, and phosphatase, while K. obovata contributed to accumulation of Cu, urease, and phosphatase. Furthermore, A. marina induced activation of Zn and accumulation of urease, phosphatase, and catalase. The characteristic enzyme and metal distributions induced by the different mangrove species are likely to result from different planting times, root systems, and soil pH and salinity. Moreover, the three mangrove species were found to influence the diversity of elemental and enzymatic stoichiometry through differences in the soil microenvironment, which can promote biodiversity in wetland ecosystems. These findings provide a useful guideline to develop strategies for restoration of estuarine wetlands.

Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme.

岷江上游干旱河谷灌丛群落土壤生态酶化学计量特征

PDF HTML阅读 XML下载 导出引用 引用提醒 岷江上游干旱河谷优势灌丛群落土壤生态酶化学计量特征 DOI: 10.5846/stxb201401270202 作者: 作者单位: 中国科学院生态环境研究中心,中国科学院生态环境研究中心城市与区域国家重点实验室,中国科学院生态环境研究中心城市与区域国家重点实验室 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金(31170581,41101270) Patterns of ecoenzymatic stoichiometry in the dominant shrubs in the semi-arid Upper Minjiang River Valley Author: Affiliation: Research Center for Eco-environmental Science, Chinese Academy of Sciences,Research Center for Eco-environmental Science, Chinese Academy of Sciences, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:与碳(C)、氮(N)和磷(P)分解矿化相关的土壤生态酶的相对活性可以揭示微生物生长和代谢过程的能量限制。尽管干旱生态系统愈来愈受到生态学家的关注,但在土壤水分有效性限制和斑块状分布的植被背景下,探讨土壤酶活性化学计量特征,及其与土壤物理、化学因子和土壤微生物群落结构的关系的相关研究仍十分缺乏。通过调查岷江干旱河谷不同水分梯度4个研究样地(两河口、飞虹、撮箕和牟托)灌丛及其丛间空地的土壤物理化学性质、微生物群落组成和土壤生态酶活性,探讨不同水分梯度样地间及灌丛和空地间土壤酶活性计量的分布特征。结果显示,岷江干旱河谷土壤C : N : P酶活性化学计量表现出了与全球尺度上酶活性化学计量的相对特异性(C : N : P = 3 : 2 : 2 vs. 1 : 1 : 1)。整体上,岷江干旱河谷区域土壤微生物生长代谢存在一定程度的N限制。虽然酶活性化学计量特征在所有空地和灌丛土壤之间总体未表现出显著差异,但对各研究地的进一步分析表明,两个相对干旱的两河口和飞虹样地表现出更为明显的N限制,而两个相对湿润的撮箕和牟托样地则存在一定的P限制。这种样地间的差异性主要受土壤水分有效性的驱动。逐步回归分析表明,土壤含水量是不同样地间土壤C : N酶活性比(ln(BG) : ln(NAG+LAP))和N : P酶活性比(ln(NAG+LAP) : ln(AP))的决定性影响因子。而土壤含水量与C : N酶活性比具有显著正相关关系,与N : P酶活性比表现为显著负相关关系。此外,土壤真菌、菌根真菌和真菌 : 细菌比对土壤酶活性化学计量的影响,说明了真菌和菌根真菌类群对干旱河谷养分循环的重要作用。 Abstract:The activities of soil ecoenzymes in relation to the mineralization of soil organic carbon, nitrogen, and phosphorus can reveal stoichiometric and energy limitations on the growth and metabolism of soil microbes. Despite the fact that arid and semi-arid ecosystems have received considerable attention in recent years, studies on the patterns of soil ecoenzymatic stoichiometry and the relationships among soil physiochemical and microbial community factors are still rare. In this study, we investigated the variations of soil physiochemical properties, microbial community composition, and EEAs by comparing soils in dominant shrubs and in the shrub-interspaces at four sites (Lianghekou, Feihong, Cuoji, and Moutuo). These study sites were expected to differ with respect to the level of water stress along the semi-arid valley in the upper Minjiang River, Sichuan province. The patterns of ecoenzymatic stoichiometry of C : N : P were different in the semi-arid Minjiang River valley as compared to those in the global pattern (C : N : P=3 : 2 : 2 vs. 1 : 1 : 1). Overall, there may be some degree of nitrogen limitation of microbial metabolism in this semi-arid region, which was supported by the relative activities of β-glucosidase compared to β-N-acetylglucosaminidase + leucine aminopeptidase and β-N-acetylglucosaminidase + leucine aminopeptidase to phosphatase. The different patterns of enzymatic stoichiometry between the soils in shrubs and in the shrub interspaces were not obvious. However, the limitation of C, N, and P resources in each individual site was somewhat site specific and may be due to the soil water availability. The two relatively drier sites (Lianghekou and Feihong) were more constrained by nitrogen availability, while the two relatively wetter sites (Cuoji and Moutuo) were more constrained by phosphorus availability. The variations of soil enzymatic stoichiometric patterns were affected by nutrient availabilities and soil water content as shown by the Pearson product-moment correlation analysis. The results of stepwise regression further revealed that the variations of enzyme ratios, such as C : N and N : P, were driven primarily by soil water content. Soil water content was positively correlated with the C : N enzyme ratio and negatively correlated with the N : P enzyme ratio. We also observed that arbuscular mycorrhizal fungi (AMF) and the fungi-to-bacteria ratio positively correlated with the C : N enzyme ratio, but negatively with the N : P enzyme ratio, which may imply that the enrichment of fungi and AMF communities will benefit C and P cycling by influencing enzyme activities. Our results suggest that any predicted decrease in soil water with climate change will probably further constrain the availability of N and P in this N-limited ecosystem in the semi-arid region. Moreover, this study also highlights the important role of fungi and AMF communities in meditating C and P cycling in this semi-arid region. 参考文献 相似文献 引证文献

Maximizing the efficiency of multienzyme process by stoichiometry optimization.

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one-pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes.

Soil aggregate size distribution mediates microbial climate change feedbacks

Soil carbon stabilization is known to depend in part on its distribution in structural aggregates, and upon soil microbial activity within the aggregates. However, the influence of climate change on continued soil C storage within aggregates of different size classes is unknown. In this study, we applied a modified dry-sieving technique to separate bulk soil into three fractions (>1 mm large macroaggregate; 0.25–1 mm small macroaggregate; <0.25 mm microaggregate), and measured the activities of seven microbial enzymes involved in the cycling of C, N, and P, in the context of a long-term elevated CO2 and warming experiment. Significant effects of aggregate size were found for most enzyme activities, enzyme stoichiometry, and specific enzyme activities (per unit of microbial biomass), suggesting that aggregate size distribution mediates microbial feedbacks to climate change. C decomposition enzyme activities, the ratios of total C:N and C:P enzyme activity, and the specific enzyme activity for C decomposition were significantly higher in the microaggregates across climate treatments. However, specific enzyme activity for N decomposition was significantly higher in macroaggregates. Increased specific enzyme activity for C decomposition under both elevated CO2 and warming suggests that these climate changes can enhance microbial ability to decompose soil organic matter (SOM). Moreover, changes in the enzyme C:N:P stoichiometry suggest that soil microorganisms may be able to adjust nutrient acquisition ratios in response to climate change. Our study suggests that identifying and modeling aggregate size as a function of SOM decomposition could improve our mechanistic understanding of soil biogeochemical cycling responses to climate change.

Seasonal patterns of microbial extracellular enzyme activities in an arctic tundra soil: Identifying direct and indirect effects of long-term summer warming

Arctic systems, which store ∼50% of global soil carbon, are undergoing rapid climatic warming that may drive significant carbon release to the atmosphere. To better understand how warming impacts arctic decomposition, we characterized the effects of a twenty-two year long tundra greenhouse warming experiment on decomposer-produced extracellular enzymes, nutrients, and microbial biomass across a year. This experiment, which is the longest running tundra ecosystem warming study in existence, was previously shown to have altered the plant and soil communities. The greenhouse treatment has also changed the seasonal soil temperature regime by indirectly increasing winter soil temperature, an effect that was likely facilitated through an increase in snow-trapping shrub biomass. Irrespective of the warming treatment, we observed that peak nutrient pools, microbial biomass, and hydrolytic enzyme activities all occurred from the late winter through thaw. This pattern was decoupled from peak oxidative enzyme activities, which occurred during the summer. The greenhouse treatment amplified the natural seasonal cycle of extracellular enzyme activities, suggesting that tundra decomposer communities maintain a temporal niche space which is critical to understanding how arctic biogeochemical cycling will respond to warming. A spatial separation was also observed; extracellular enzyme activities in the deeper soil horizons were more sensitive to warming than at the surface. Direct greenhouse warming did not strongly stimulate decomposition: only oxidative enzyme activities in the surface horizon increased during the summer. Unexpectedly, the strongest treatment effect observed was a stimulation of hydrolytic enzyme activities at depth in the mineral horizon from the late winter through thaw (which also affected extracellular enzyme stoichiometry, increasing C:N and C:P acquisition activities), before the greenhouse treatment was directly active. This effect declined during senescence and was reversed in early winter, suggesting that negative biotic-abiotic feedbacks may curtail increased decomposer activity in warming arctic systems.

Stoichiometric modeling of oxidation of reduced inorganic sulfur compounds (Riscs) in Acidithiobacillus thiooxidans

The prokaryotic oxidation of reduced inorganic sulfur compounds (RISCs) is a topic of utmost importance from a biogeochemical and industrial perspective. Despite sulfur oxidizing bacterial activity is largely known, no quantitative approaches to biological RISCs oxidation have been made, gathering all the complex abiotic and enzymatic stoichiometry involved. Even though in the case of neutrophilic bacteria such as Paracoccus and Beggiatoa species the RISCs oxidation systems are well described, there is a lack of knowledge for acidophilic microorganisms. Here, we present the first experimentally validated stoichiometric model able to assess RISCs oxidation quantitatively in Acidithiobacillus thiooxidans (strain DSM 17318), the archetype of the sulfur oxidizing acidophilic chemolithoautotrophs. This model was built based on literature and genomic analysis, considering a widespread mix of formerly proposed RISCs oxidation models combined and evaluated experimentally. Thiosulfate partial oxidation by the Sox system (SoxABXYZ) was placed as central step of sulfur oxidation model, along with abiotic reactions. This model was coupled with a detailed stoichiometry of biomass production, providing accurate bacterial growth predictions. In silico deletion/inactivation highlights the role of sulfur dioxygenase as the main catalyzer and a moderate function of tetrathionate hydrolase in elemental sulfur catabolism, demonstrating that this model constitutes an advanced instrument for the optimization of At. thiooxidans biomass production with potential use in biohydrometallurgical and environmental applications.

Microbial enzyme stoichiometry and nutrient limitation in US streams and rivers

We analyzed water and sediment chemistry, catchment land cover, and microbial dehydrogenase (DHA) and extracellular enzymes activities (EEA) related to microbial C, N, P, and S acquisition in more than 2100 1st–10th order streams. The streams and their catchments represented gradients in water and sediment chemistry (C, N, P, S) and land cover (% forest, % wetland, % row crop agriculture) against which to compare biofilm and sediment DHA and EEA, and to estimate the extent of nutrient limitation in US streams and rivers. Water chemistry was significantly correlated with catchment land cover. Biofilm and sediment DHA and EEA were inversely correlated with water and sediment chemistry, and positively correlated with % forest. Canonical correlation analysis described two environmental gradients to which biofilm and sediment DHA and EEA were significantly correlated. Structural equation modeling (SEM) revealed a significant causal relationship between catchment land cover, chemistry, and biofilm and sediment EEA and DHA. Biofilm and sediment EEA was dominated by phenol oxidase and peroxidase, enzymes related to the degradation of recalcitrant C. However, the hydrolytic enzymes (those which hydrolyze glycoside, peptide, and ester bonds to release C, N, P, S) were better correlated with the chemical variables. This was reflected in the relative apportionment of enzyme activity toward C, N, P, and S acquisition. We compared molar stoichiometry of C, N, and P in water and sediment with biofilm and sediment ratios of GLYC:PEPT:PHOS to gauge how each measure of nutrient limitation compares with the others. Overall, water chemistry suggested many biofilms were P-limited and this was corroborated by allocation of microbial phosphatase activity. Sediments were found to be predominantly C and N-limited, and this was reflected in sediment EEA. We used these measures of nutrient limitation to assess the extent of N or P-limitation in streams and rivers of the conterminous United States and for nine ecoregions of interest to the US EPA's National Rivers and Streams Assessment (NRSA). Enzymatic stoichiometry provides a biological perspective on the influence of catchment scale anthropogenic disturbances resulting in an imbalance of nutrients being transported from those catchments. Enzyme activities represent the interface between microbial demands for, and environmental supplies of, C, N, and P, effectively linking ecological stoichiometric theory with the concept of threshold elemental ratios. The relative activities of the functional classes of extracellular enzymes provide both a measure of nutrient availability and ecosystem metabolism that may be used to assess large-scale phenomena such as regional impacts of climate change or anthropogenic disturbances.

Effects of land use on water column bacterial activity and enzyme stoichiometry in stream ecosystems

Fifty streams, located in southern Ontario, Canada, were visited in September 2008 to investigate the effect of varying land use, land cover, and associated resource inputs on water column bacterial abundance (BACT), production (BP), and extracellular enzyme activity and stoichiometry. Principle components analysis was used to summarize landscape data, producing three components (PCs), which explained 79.2% of the variability in the data. The PCs grouped into the following gradients: (PC1) urban land use and continuous annual cropping to wetland-like cover, (PC2) rotational cropping to forest-like cover, and (PC3) increasing rural and agricultural land uses with increasing watershed size. These landscape gradients created imbalanced resource availability. Nutrient resources were more abundant in streams with more intensive anthropogenic land uses, but carbon availability was primarily controlled by the abundance of natural land covers (wetland and wooded areas). BACT, BP, and enzyme activities were positively related primarily to nutrient availability and/or anthropogenic land use (Stepwise R 2 range: 0.33–0.73). The ratio of β-glucosidase to alkaline phosphatase activity approached a 1:1 balance with increasing anthropogenic land use, decreased wetland and forest cover, and increased total dissolved nitrogen. The ratio of leucine-aminopeptidase to alkaline phosphatase activity approached 1:1 with both increased dissolved organic carbon and nitrogen. Moreover, enzyme C:N:P ratios moved closer to 1:1:1 with faster water column bacterial turnover times. These results suggest that water column microbial communities are better able to balance resource availability with growth in streams receiving nutrient subsidies from anthropogenic sources and under these conditions when carbon resources increase.

Extracellular enzyme activity in the mycorrhizospheres of a boreal fire chronosequence

Abstract Saprotrophic microbes are typically credited with producing extracellular enzymes that recycle organic matter, though roots and mycorrhizal fungi also can contribute and may compete with the saprotrophs. We examined extracellular enzyme activity associated with the mycorrhizospheres of arbuscular mycorrhizal, ectomycorrhizal, dual-colonized (arbuscular and ectomycorrhizal), and ericoid mycorrhizal plants in a fire chronosequence in Alaska. Bulk soil and soil from beneath host plants were gathered in July 2004 and assayed for five enzymes that target organic C, P, and N substrates. Compared to bulk soil, activities of the C-targeting enzymes β-1,4-glucosidase and peroxidase were lower in arbuscular mycorrhizospheres and ericoid mycorrhizospheres, respectively. Moreover, extracellular enzyme activity varied among mycorrhizosphere types. Specifically, N-targeting leucine aminopeptidase was highest in arbuscular mycorrhizospheres, followed by ericoid and ectomycorrhizal/dual-colonized mycorrhizospheres; β-1,4-glucosidase had the reverse pattern. In addition, enzymatic stoichiometry suggested that extracellular enzyme producers invested more in C-acquisition than in N-acquisition in recent fire scars compared to mature forests. These data extend previous findings that roots and mycorrhizal fungi compete with saprotrophs by showing that the strength of this effect varies by mycorrhizal host. As a result the community composition of mycorrhizal host plants might mediate enzymatic activity in boreal soils.

Stoichiometry of Base Excision Repair Proteins Correlates with Increased Somatic CAG Instability in Striatum over Cerebellum in Huntington's Disease Transgenic Mice

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5′-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5′-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP–BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLβ was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLβ strand displacement activity during LP–BER promotes the formation of stable 5′-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical factor underlying the tissue selectivity of somatic CAG expansion.

Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors.

Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a "critical aggregation concentration" (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10,000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 x 10(11) and 4.7 x 10(10) A(3), respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate.

Oxidation of Inorganic Sulfur Compounds in Acidophilic Prokaryotes

AbstractThe oxidation of reduced inorganic sulfur compounds to sulfuric acid is of great importance for biohydrometallurgical technologies as well as the formation of acidic (below pH 3) and often heavy metal‐contaminated environments. The use of elemental sulfur as an electron donor is the predominant energy‐yielding process in acidic natural sulfur‐rich biotopes but also at mining sites containing sulfidic ores. Contrary to its significant role in the global sulfur cycle and its biotechnological importance, the microbial fundamentals of acidophilic sulfur oxidation are only incompletely understood. Besides giving an overview of sulfur‐oxidizing acidophiles, this review describes the so far known enzymatic reactions related to elemental sulfur oxidation in acidophilic bacteria and archaea. Although generally similar reactions are employed in both prokaryotic groups, the stoichiometry of the key enzymes is different. Bacteria oxidize elemental sulfur by a sulfur dioxygenase to sulfite whereas in archaea, a sulfur oxygenase reductase is used forming equal amounts of sulfide and sulfite. In both cases, the activation mechanism of elemental sulfur is not known but highly reactive linear sulfur forms are assumed to be the actual substrate. Inhibition as well as promotion of these biochemical steps is highly relevant in bioleaching operations. An efficient oxidation can prevent the formation of passivating sulfur layers. In other cases, a specific inhibition of sulfur biooxidation may be beneficial for reducing cooling and neutralization costs. In conclusion, the demand for a better knowledge of the biochemistry of sulfur‐oxidizing acidophiles is underlined.

Ecotin modulates thrombin activity through exosite-2 interactions

Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human α-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin–α-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC 50 = 3.2 μM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC 50 = 4.5 μM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-α-thrombin (EC 50 = 3.5 μM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.