Distinct Enzymes Research Articles

Activity-based probes for functional interrogation of retaining β-glucuronidases.

Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality.

Crystal structure of tubulin tyrosine ligase-like 3 reveals essential architectural elements unique to tubulin monoglycylases

Glycylation and glutamylation, the posttranslational addition of glycines and glutamates to genetically encoded glutamates in the intrinsically disordered tubulin C-terminal tails, are crucial for the biogenesis and stability of cilia and flagella and play important roles in metazoan development. Members of the diverse family of tubulin tyrosine ligase-like (TTLL) enzymes catalyze these modifications, which are part of an evolutionarily conserved and complex tubulin code that regulates microtubule interactions with cellular effectors. The site specificity of TTLL enzymes and their biochemical interplay remain largely unknown. Here, we report an in vitro characterization of a tubulin glycylase. We show that TTLL3 glycylates the β-tubulin tail at four sites in a hierarchical order and that TTLL3 and the glutamylase TTLL7 compete for overlapping sites on the tubulin tail, providing a molecular basis for the anticorrelation between glutamylation and glycylation observed in axonemes. This anticorrelation demonstrates how a combinatorial tubulin code written in two different posttranslational modifications can arise through the activities of related but distinct TTLL enzymes. To elucidate what structural elements differentiate TTLL glycylases from glutamylases, with which they share the common TTL scaffold, we determined the TTLL3 X-ray structure at 2.3-Å resolution. This structure reveals two architectural elements unique to glycyl initiases and critical for their activity. Thus, our work sheds light on the structural and functional diversification of TTLL enzymes, and constitutes an initial important step toward understanding how the tubulin code is written through the intersection of activities of multiple TTLL enzymes.

First Report of a 16SrXIV Group Phytoplasma Associated With Witches’-Broom Disease of Acid Lime (Citrus aurantifolia) in India

HomePlant DiseaseVol. 101, No. 5First Report of a 16SrXIV Group Phytoplasma Associated With Witches’-Broom Disease of Acid Lime (Citrus aurantifolia) in India PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a 16SrXIV Group Phytoplasma Associated With Witches’-Broom Disease of Acid Lime (Citrus aurantifolia) in IndiaD. K. Ghosh, S. Bhose, P. Sharma, A. Warghane, M. Motghare, M. S. Ladaniya, M. K. Reddy, V. Thorat, and A. YadavD. K. GhoshSearch for more papers by this author, S. BhoseSearch for more papers by this author, P. SharmaSearch for more papers by this author, A. WarghaneSearch for more papers by this author, M. MotghareSearch for more papers by this author, M. S. LadaniyaSearch for more papers by this author, M. K. ReddySearch for more papers by this author, V. ThoratSearch for more papers by this author, and A. YadavSearch for more papers by this authorAffiliationsAuthors and Affiliations D. K. Ghosh S. Bhose P. Sharma A. Warghane M. Motghare M. S. Ladaniya , ICAR-Central Citrus Research Institute, Nagpur-440 033, India M. K. Reddy , ICAR-Indian Institute of Horticultural Research, Bangalore-560 089, India V. Thorat A. Yadav , Microbial Culture Collection, National Center for Cell Science, Pune - 411 021, India. Published Online:9 Mar 2017https://doi.org/10.1094/PDIS-11-16-1549-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Acid lime [Citrus aurantifolia (L.) Swingle] is an important citrus cultivar that constitutes nearly 20% of total citrus production in India. During a survey conducted in April 2014 to February 2015, conspicuous symptoms of small chlorotic leaves with highly proliferated shoots and shortened internodes resembling witches’-broom disease of acid lime (WBDL) were observed in four out of 22 acid lime groves in Nagpur district of Maharashtra state, India. Seven representative field samples from symptomatic plants were graft-inoculated to healthy acid lime seedlings under greenhouse conditions and all developed characteristic symptoms 18 to 20 months after grafting. Numerous phytoplasma-like bodies of 100 to 800 nm in diameter and bounded by poorly defined membrane were observed in sieve tubes of diseased samples but not in similar sections from healthy leaves when ultrathin sections of leaf midrib were observed under a JEM 100S transmission electron microscope. PCR was used to confirm the presence of phytoplasma in infected tissues from 20 symptomatic and five asymptomatic plants. Samples were analyzed using P1/P7 primer pairs for the first round of PCR and the products were subsequently amplified with nested primer pairs R16F2n/R16R2 (Lee et al. 2004). An expected amplicon of ∼1.2 kb was obtained from 18 diseased plant tissues while it was not observed in asymptomatic plants. Four amplicons obtained from different field samples using R16F2n/R16R2 were purified, sequenced, and the resultant sequences were deposited in GenBank (KY024345, KY024346, KY024347, KY024348). All obtained sequences were 100% identical. The results of NCBI-BLAST analysis further revealed that the 16S rRNA gene sequences shared 99.9% similarity with that of the ‘Candidatus Phytoplasma cynodontis’ strains (LT558777, KF234570, AB741630). Phylogenetic analysis of obtained sequences showed that the WBDL phytopalsma formed a distinct clade with ‘Ca. Phytoplasma cynodontis’ strain BGWL-C1. In silico RFLP analysis of the aligned 16S rRNA gene sequence of the WBDL phytoplasma was performed with 17 distinct restriction enzymes using iPhyClassifier (Zhao et al. 2009) and the results were compared with each phytoplasma group and subgroup. Based on the analysis, WBDL phytoplasma 16S rRNA gene sequences shared 99.8% similarity with that of the ‘Ca. Phytoplasma cynodontis’ reference strain (AJ550984). Moreover, the virtual RFLP pattern derived from the query 16S rRNA gene F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XIV, subgroup A (AJ550984) (Marcone et al. 2004). Taken together, these results indicate that the phytoplasma associated with WBDL in India is a ‘Ca. Phytoplasma cynodontis’-related strain and belongs to 16SrXIV-A subgroup. To the best of our knowledge, this is the first report of occurrence of 16SrXIV group phytoplasma associated with WBDL not only in India but also in the world. It will be interesting to identify potential plant reservoir and insect vectors of the pathogen in this region. The disease adversely affects fruit yield and has the potential to significantly reduce economic condition of citrus growers of the region.

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2.

Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.

Two distinct periplasmic enzymes are responsible for tellurite/tellurate and selenite reduction by strain ER-Te-48 associated with the deep sea hydrothermal vent tube worms at the Juan de Fuca Ridge black smokers.

Strain ER-Te-48 isolated from a deep-ocean hydrothermal vent tube worm is capable of resisting and reducing extremely high levels of tellurite, tellurate, and selenite, which are used for respiration anaerobically. Tellurite and tellurate reduction is accomplished by a periplasmic enzyme of 215kDa comprised of 3 subunits (74, 42, and 25kDa) in a 2:1:1 ratio. The optimum pH and temperature for activity is 8.0 and 35°C, respectively. Tellurite reduction has a V max of 5.6µmol/min/mg protein and a K m of 3.9mM. In the case of the tellurate reaction, V max and K m were 2.6µmol/min/mg protein and 2.6mM, respectively. Selenite reduction is carried out by another periplasmic enzyme with a V max of 2.8µmol/min/mg protein, K m of 12.1mM, and maximal activity at pH 6.0 and 38°C. This protein is 165kDa and comprised of 3 subunits of 98, 44, and 23kDa in a 1:1:1 ratio.

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast

The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ∼20-60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ∼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage.

Effects of benzo[a]pyrene dietary intake to antioxidative enzymes of Lymantria dispar (Lepidoptera: Lymantriidae) larvae from unpolluted and polluted forests

Anthropogenic activity in industrial development has imposed great threats to the environment and wildlife in the form of persistent organic pollutants. Polycyclic aromatic hydrocarbons (PAH) tend to accumulate in vegetation foliage which is the main food source of polyphagous insect species Lymantria dispar L. Origin and multigenerational adaptation of L. dispar population to environmental challenges strongly condition the enzymes’ sensitivity to pollutants. In this study, our aim was to investigate response of the superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) to the chronic dietary exposure of benzo[a]pyrene in the midgut tissues and hemolymph of two L. dispar populations originating from unpolluted and polluted forest habitat. Midgut tissue of the larvae from the polluted forest showed significant increase in SOD, CAT and GST activity, while in unpolluted forest’s larvae SOD and CAT showed elevated activities in hemolymph. L. dispar populations adapted to different level of pollution in their environment and expressed distinct tissue-dependent antioxidative enzyme sensitivity to benzo[a]pyrene diet, implying high potential for further elucidation of these enzymes as molecular biomarkers.

Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh housekeeping gene of mammal hosts.

BackgroundThe PCR assays usually employed for Leishmania diagnosis does not simultaneously detect a constitutive gene that would certify the viability of the DNA sample. We present a multiplex PCR approach for the simultaneous diagnosis of the Leishmania sp. kDNA fragment and a catalytic domain segment of a conserved region of the mammalian gapdh gene.MethodologyThe proposed multiplex protocol was designed through in silico PCR. The annealing temperature, concentration of primer pairs, number of cycles, distinct polymerase enzymes and premix kit were defined to achieve an optimal reaction. The DNA detection sensitivity was tested with different concentrations of known L. tropica DNA, and the reproducibility of the assay was confirmed using samples from 106 wild mammals that were previously subject to Leishmania sp. kDNA analysis through singleplex reactions.Principal findingsThe following optimal conditions were established: 95°C for 1 min followed by 30 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 1 min. The multiplex PCR system was capable of detecting 0.1 ng of L. tropica diluted in 100 ng of mammalian DNA. Of 51 kDNA samples that were previously found to be positive, 45 (88.2%) were positive for both targets, two were positive only for kDNA and four were negative for both. Of 55 kDNA samples that were previously identified as negative, 38 (69.1%) were positive for gapdh whereas the other 17 were negative for both targets.Conclusions/SignificanceThe proposed multiplex PCR system allows the simultaneous detection of the gapdh gene and Leishmania sp. kDNA in tissue samples derived from distinct wild mammal species. The amplification of the gapdh mammalian gene in the same reaction ensures the quality and viability of the DNA in the sample, eliminating the possibility of false-negative results that would impair an accurate description of the infection rates in a given population.

Interstrain differences in the expression and activity of Cyp2a5 in the mouse liver

BackgroundCytochrome P450 2A5 (Cyp2a5), a mouse enzyme orthologous of human CYP2A6, catalyzes a number of toxicologically important reactions, including the metabolism of nicotine, aflatoxin B1, and several other xeno- and endobiotics. Cyp2a5 expression is complex and not yet fully understood. We investigated inter-strain differences in the activity and mRNA expression of hepatic Cyp2a5. Cyp1a1/2 and Cyp2b9/10 activities were evaluated for comparative purposes. Data on the interstrain differences in the expression and activity of Cyp2a5 are important to select a suitable mouse model for studying CYP2A6-mediated metabolism.ResultsActivity of Cyp2a5 (coumarin 7-hydroxylase) was highest in DBA-2 and DBA-1, intermediate in B6D2F1 (hybrid) and low in the remaining strains (C57BL/6, C57BL/10, CBA, BALB/cAn, SW). Contrasting with the activity, background levels of Cyp2a4/5 mRNA did not differ between high- and low-activity murine strains. Phenobarbital (PB, 80 mg/kg body weight/day × 3 days, i.p.) increased Cyp2a5, Cyp1a1/2 (ethoxyresorufin-O-deethylase) and Cyp2b9/10 (bezyloxyresorufin-O-debenzylase) activities while only Cyp2a5 was enhanced by pyrazole (PYR, 100 mg/kg body weight/day × 3 days, i.p.). Inductions of Cyp2a5 activity by PYR and PB were accompanied by increases of Cyp2a4/5 mRNA. PYR and PB did not upregulate heme oxygenase-1 (hmox-1) mRNA expression in any strain, a finding that is apparently at odds with the notion that Cyp2a5 and hmox-1 inductions are coordinated events.ConclusionsSince background levels of Cyp2a4/5 gene transcripts of high-activity strains did not differ from those of low-activity mice, distinct constitutive activities did not result from different transcription rates and/or mRNA half-lives. Results therefore suggested that interstrain differences in constitutive activity of Cyp2a5 possibly arise from distinct translation efficiencies, protein half-lives and/or enzyme kinetics toward the substrate. Data from this study indicated that all tested strains are suitable models for studying toxicants that are substrates for human CYP2A6; DBA-2, DBA-1 and the hybrid B62DF1, however, have the advantage of presenting high constitutive activities of Cyp2a5.

Cellular Dynamics Controlled by Phosphatases

Protein phosphorylation, a fundamental post-translation modification Post-translation modification: Post-translational modification is covalent modification of proteins that occur during or after protein synthesis, where functional groups are added by enzymatic action. that acts as a backbone of signaling networks, is essential for multiple aspects of eukaryote physiology. Phosphorylation status of a substrate is dependent on opposing activities of two distinct enzymes, where the relevant kinase catalyzes the modification and is reversed by a phosphatase Phosphatase: Enzymes that remove phosphate group from substrates by hydrolysis. . Historically, kinases have been at the research forefront; however, phosphatases have gained importance with many studies revealing predominant roles for these enzymes in controlling the cellular responses. Phosphatases are known to attenuate or amplify signaling by operating both as early, as well as delayed regulators of signal transduction. This review is focused on describing the versatile roles of phosphatases in controlling different cellular pathways through their spatio-temporal dynamics during signaling.

Innate function of house dust mite allergens: robust enzymatic degradation of extracellular matrix at elevated pH

Background Exposure to the house dust mite Dermatophagoides pteronyssinus (D.p.) increases the risk for developing allergic diseases in humans and their best friends, the dogs. Here, we explored whether this allergenic mite via its enzymes may impact the cutaneous extracellular matrix (ECM), which critically determines epithelial barrier integrity both structurally and functionally.Methods Two extracts obtained from either dust-purified or cultured D.p. bodies were used in the present study. To assess the potential impact of D.p. on protein components of the ECM, proteolytic activity of the D.p. extracts were determined by casein and gelatin gel zymography, and their N-acetyl-β-hexosaminidase activity determined colorimetrically. In addition, IgE-dependent and innate degranulation potential of D.p. was examined in canine MPT-1 mast cells and neurite outgrowth assay using rat pheochromocytoma PC-12 cells.Results In gel zymography, both extracts digested the substrates casein and gelatin in a dose-dependent manner, especially at alkaline pH, and effective in a wide range of temperatures (30 °C−42 °C). In particular, a 25-kDa band corresponding to Der p 1, the major D.p. allergen for humans, was found enzymatically active in both casein and gelatin gels regardless of the presence of metal ions and of alkaline conditions. Besides protease activity, N-acetyl-β-hexosaminidase activity was detected in both extracts, suggesting that D.p. affects the cutaneous ECM through deteriorating both proteins and glycosaminoglycans. While both D.p. extracts induced IgE-dependent mast cell degranulation, much less innate effects on mast- and neuronal cells were observed.Conclusions Our data highlight that D.p. is a robust source of several distinct enzymes with protease- and N-acetyl-β-hexosaminidase activities. In alkaline milieu they can degrade components of the ECM. Therefore, D.p. may contribute to epithelial barrier disruption especially when the skin surface pH is elevated.

Abstract IA23: Enhancing anti-tumor immunity through selective inhibition of IDO1

Abstract Tryptophan catabolism serves as an important metabolic immunoregulatory checkpoint, functioning in anti-inflammatory, immunosuppressive and tolerance-inducing processes. Three distinct heme-containing enzymes—indoleamine 2,3-dioxygenase (IDO) 1, IDO2 and tryptophan 2,3-dioxygenase (TDO)—have the capacity to initiate the first and rate-limiting step in this metabolic process, catalyzing the conversion of tryptophan to kynurenine. A growing body of scientific evidence indicates that this pathway, and more specifically, the IDO1 enzyme, may be of fundamental importance to tumor immune evasion. Through the depletion of local tryptophan and the production of kynurenine and other downstream metabolites, IDO1 activity has been shown to have an anti-proliferative effect on T cells, to promote Treg differentiation and to bias dendritic cells and macrophages toward an immunosuppressive phenotype. Further, IDO1 expression is a negative prognostic factor across numerous cancer histologies. Therefore, the selective inhibition of IDO1 has emerged as an attractive therapeutic approach to enhance anti-tumor immunity. This presentation will review the immunologic basis for targeting tryptophan catabolism in cancer, describe the emerging preclinical and clinical data on epacadostat, a novel first-in-class selective IDO1 inhibitor, and discuss the therapeutic rationales for epacadostat-based combination immunotherapies. Citation Format: Reid Huber. Enhancing anti-tumor immunity through selective inhibition of IDO1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA23.

Structural Characterization of Poised States in the Oxygen Sensitive Hydrogenases and Nitrogenases.

The crystallization of FeS cluster-containing proteins has been challenging due to their oxygen sensitivity, and yet these enzymes are involved in many critical catalytic reactions. The last few years have seen a wealth of innovative experiments designed to elucidate not just structural but mechanistic insights into FeS cluster enzymes. Here, we focus on the crystallization of hydrogenases, which catalyze the reversible reduction of protons to hydrogen, and nitrogenases, which reduce dinitrogen to ammonia. A specific focus is given to the different experimental parameters and strategies that are used to trap distinct enzyme states, specifically, oxidants, reductants, and gas treatments. Other themes presented here include the recent use of Cryo-EM, and how coupling various spectroscopies to crystallization is opening up new approaches for structural and mechanistic analysis.

Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2.

Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe's target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (Ki = 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP.

Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation

The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.

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.

DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target.

R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5΄-ATTTAAAT-3΄, and cleaves that target at its center to generate blunt-ended DNA fragments. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions; and a DNA-bound, fully cleaved complex with magnesium ions. We compare these structures to two structurally similar ‘PD-D/ExK’ restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage.

Cell biology: Double agents for mitochondrial division.

Mitochondrial organelles — the energy powerhouses of the cell — must divide and fuse dynamically to function. It emerges that two distinct dynamin enzymes enable mitochondrial division. See Letter p.139 Mitochondria—the cellular organelles responsible for respiration and energy production—are dynamic systems that continuously undergo fission and fusion. It has been assumed that mitochondrial division, which involves remodelling of the mitochondrial membranes and constriction from about 500 nanometres down to 10 nanometres, is performed entirely by Drp1 (mitochondrial dynamin-related protein 1). Gia Voeltz and colleagues now report that, in addition to Drp1, mammalian cells use the classical dynamin-2 (Dyn2) to further constrict and divide mitochondria. In a sequential process, just prior to division Dyn2 seems to localize to mitochondrial constriction sites that have already been remodelled by Drp1.

Overlapping Role of Respiratory Supercomplex Factor Rcf2 and Its N-terminal Homolog Rcf3 in Saccharomyces cerevisiae

The mitochondrial electron transport chain consists of individual protein complexes arranged into large macromolecular structures, termed respiratory chain supercomplexes or respirasomes. In the yeast Saccharomyces cerevisiae, respiratory chain supercomplexes form by association of the bc1 complex with the cytochrome c oxidase. Formation and maintenance of these assemblies are promoted by specific respiratory supercomplex factors, the Rcf proteins. For these proteins a regulatory function in bridging the electron transfer within supercomplexes has been proposed. Here we report on the maturation of Rcf2 into an N- and C-terminal peptide. We show that the previously uncharacterized Rcf3 (YBR255c-A) is a homolog of the N-terminal Rcf2 peptide, whereas Rcf1 is homologous to the C-terminal portion. Both Rcf3 and the C-terminal fragment of Rcf2 associate with monomeric cytochrome c oxidase and respiratory chain supercomplexes. A lack of Rcf2 and Rcf3 increases oxygen flux through the respiratory chain by up-regulation of the cytochrome c oxidase activity. A double gene deletion of RCF2 and RCF3 affects cellular survival under non-fermentable growth conditions, suggesting an overlapping role for both proteins in the regulation of the OXPHOS activity. Furthermore, our data suggest an association of all three Rcf proteins with the bc1 complex in the absence of a functional cytochrome c oxidase and identify a supercomplex independent interaction network of the Rcf proteins.

Molecular docking studies of dithiocarbamates compounds interactions with jack-bean urease

Abstract Molecular docking studies concerning the inclusion of dithiocarbamates compounds inside the jack-bean urease enzyme (JBU) are reported. The compounds are dimethyldithiocarbamate (MDTC), diethyldithiocarbamate (EDTC) and pyrrolidinedithiocarbamate (PDTC) and they present inhibitory activity against urease, justifying the structural investigation. Interaction energies of each dithiocarbamate inside the distinct enzyme domains are calculated and the active sites for the inhibitors are determined. The lowest electrostatic intermolecular energy (ΔE) for every compound was obtained within the C-terminal (αβ) 8 domain containing the Tyr442 residue, originally corresponding to the enzymatic catalytic site. EDTC and PDTC compounds have similar ΔE and MDTC has a comparatively higher ΔE . It was observed that the stability of inclusion increases with the carbon chain of the molecules and from the ΔE perspective the relative inhibitors efficiencies are in order PDTC ≈ EDTC > MDTC. However, interactions of the dithiocarbamates with water molecules allowed determining a precise order of activity as EDTC > PDTC > MDTC. Data are supplied in this article.