Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k_cat/K_m values >10,000 M^?1 s^?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k_cat/K_m values exhibited against these canonical substrates are well under 10⁵ M^?1 s^?1. By contrast, several Nudix enzymes show much larger k_cat/K_m values (in the range of 10⁵ to >10⁷ M^?1 s^?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Potential of Ulocladium atrum for biocontrol of onion leaf spot through suppression of sporulation of Botrytis spp.

Epidemics of onion leaf spotcaused by Botrytis spp. depend onnecrotic leaf tissue for inoculum build up inthe crop. Ulocladium atrum Preuss. is astrong competitor on necrotic above-groundplant tissues. The potential of the antagonistto reduce colonisation of necrotic leaf tissueby Botrytis spp. and subsequentsporulation was studied in two fieldexperiments. U. atrum colonised necrotictissues and consistently reduced thesporulation of fungal competitors. Althoughincidence of Botrytis spp. was low,significantly lower spore loads of Botrytis spp. were found on spore trapslocated within U. atrum treated onionplots as compared to untreated control plots.The number of leaf spots caused by Botrytis spp. was low in both fieldexperiments but was significantly reduced by60% after U. atrum applications in thesecond experiment. Spraying of the fungicideRonilan resulted in the same control level.Results show that U. atrum has apotential for biological control against diseases caused by Botrytis spp. in onions.     

Alteration of substrate specificity of valine dehydrogenase from Streptomyces albus

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide and polyether antibiotic biosyntheses. To determine the active site residues of ValDH, we previously cloned, partially characterized, and identified the active site (lysine) of Streptomyces albus ValDH. Here we report further characterization of S. albus ValDH. The molecular weight of S. albus ValDH was determined to be 38 kDa by SDS-PAGE and 67 kDa by gel filtration chromatography indicating that the enzyme is composed of two identical subunits. Optimal pHs were 10.5 and 8.0 for dehydrogenase activity with valine and for reductive amination activity with -ketoisovaleric acid, respectively. Several chemical reagents, which modify amino-acid side chains, inhibited the enzyme activity. To examine the role played by the residue for enzyme specificity, we constructed mutant ValDH by substituting alanine for glycine at position 124 by site-directed mutagenesis. This residue was chosen because it has been considered to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). The Ala-124–Gly mutant enzyme displayed lower activities toward aliphatic amino acids, but higher activities toward L-phenylalanine, L-tyrosine, and L-methionine compared to the wild type enzyme suggesting that Ala-124 is involved in substrate binding in S. albus ValDH.     

Formation of the exopolysaccharide ethapolan by <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. IMV B-7005 on a fumarate-glucose mixture

Our studies enabled us to intensify the synthesis of the microbial exopolysaccharide (EPS) ethapolan produced by Acinetobacter sp. IMV B-7005 grown on a mixture of fumarate (an energy-excessive substrate) and glucose (an energy-deficient substrate). Supplementing glucose-containing medium with sodium (potassium) fumarate at a molar ratio of 4: 1 resulted in a 1.3–2.2-fold increase of the EPS amount synthesized and in a 1.3–2-fold increase of the EPS yield relative to the biomass compared to cultivation on monosubstrates. The conversion of the carbon of both substrates to EPS was the highest if the carbon/nitrogen ratio in the cultivation medium was 70.5 and inoculum grown on glucose monosubstrate was used.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology

Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase chain reaction (PCR)-amplified fluorescently labeled DNA extracted from pure culture and from diseased plant tissue. The probes easily distinguished these pathogens from each other without cross reaction. Thickness of the agarose layer and length of the sample DNA were important factors affecting hybridization efficiency of immobilized probe to PCR product. These factors did not affect hybridization with short complementary oligonucleotide. Probes fixed on agarose-coated slides could differentiate samples as readily as probes on nylon but with potentially higher spot density and gave much better signal than probes on silylated slides. The use of plain glass slides, agarose, and a manual arrayer makes this technique useful for developing specialized and inexpensive DNA microarrays on a solid rigid substrate.     

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

The regulation of caspase‐3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase‐3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5‐P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub‐site by increasing flexibility of one active site loop and by affecting hydrogen‐bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase‐6 in zebrafish development.     

Technical note: Tree truthing: How accurate are substrate estimates in primate field studies?

Field studies of primate positional behavior typically rely on ground‐level estimates of substrate size, angle, and canopy location. These estimates potentially influence the identification of positional modes by the observer recording behaviors. In this study we aim to test ground‐level estimates against direct measurements of support angles, diameters, and canopy heights in trees at La Suerte Biological Research Station in Costa Rica. After reviewing methods that have been used by past researchers, we provide data collected within trees that are compared to estimates obtained from the ground. We climbed five trees and measured 20 supports. Four observers collected measurements of each support from different locations on the ground. Diameter estimates varied from the direct tree measures by 0–28 cm (Mean: 5.44 ± 4.55). Substrate angles varied by 1–55° (Mean: 14.76 ± 14.02). Height in the tree was best estimated using a clinometer as estimates with a two‐meter reference placed by the tree varied by 3–11 meters (Mean: 5.31 ± 2.44). We determined that the best support size estimates were those generated relative to the size of the focal animal and divided into broader categories. Support angles were best estimated in 5° increments and then checked using a Haglöf clinometer in combination with a laser pointer. We conclude that three major factors should be addressed when estimating support features: observer error (e.g., experience and distance from the target), support deformity, and how support size and angle influence the positional mode selected by a primate individual. individual. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.     

土壤呼吸温度敏感性的影响因素和不确定性

土壤呼吸是陆地生态系统碳循环的重要环节之一, 其对温度升高的敏感程度在很大程度上决定着全球气候变化与碳循环之间的反馈关系。为了深刻理解地下生态过程对气候变化的响应和适应,本文综述了土壤呼吸温度敏感性（Q10）的影响因子及其内在机制,并分析了当前研究存在的不确定性。土壤生物、底物质量和底物供应显著调控着土壤呼吸的Q10值,但研究结论仍然有很大差异。温度和水分等环境因子则通过对土壤生物和底物的影响而作用于土壤呼吸的温度敏感性,一般情况下,随着温度的升高,土壤呼吸的Q10值下降;水分过高或过低时Q10值降低。另外本文从土壤温度测定深度、时空尺度、土壤呼吸不同组分温度敏感性差异、激发效应以及采用方法的不同等几方面分析了温度敏感性研究存在的不确定性。并在此基础上, 指出了未来拟重点加强的研究方向:（1）土壤呼吸不同组分温度敏感性差异的机理;（2）底物质量和底物供应对温度敏感性的交互影响;（3）生物因子对土壤呼吸温度敏感性的影响。     

PEGDA hydrogels with patterned elasticity: Novel tools for the study of cell response to substrate rigidity

The ability of cells to migrate in response to mechanical gradients (durotaxis) and differential cell behavior in adhesion, spreading, and proliferation in response to substrate rigidity are key factors both in tissue engineering, in which materials must be selected to provide the appropriate mechanical signals, and in studies of mechanisms of diseases such as cancer and atherosclerosis, in which changes in tissue stiffness may inform cell behavior. Using poly(ethylene glycol) diacrylate hydrogels with varying polymer chain length and photolithographic patterning techniques, we are able to provide substrates with spatially patterned, tunable mechanical properties in both gradients and distinct patterns. The hydrogels can be patterned to produce anisotropic structures and exhibit patterned strain under mechanical loading. These hydrogels may be used to study cell response to substrate rigidity in both two and three dimensions and can also be used as a scaffold in tissue‐engineering applications. Biotechnol. Bioeng. 2010; 105: 636–644. © 2009 Wiley Periodicals, Inc.     

Crius: A novel fragment‐based algorithm of de novo substrate prediction for enzymes

The study of enzyme substrate specificity is vital for developing potential applications of enzymes. However, the routine experimental procedures require lot of resources in the discovery of novel substrates. This article reports an in silico structure‐based algorithm called Crius, which predicts substrates for enzyme. The results of this fragment‐based algorithm show good agreements between the simulated and experimental substrate specificities, using a lipase from Candida antarctica (CALB), a nitrilase from Cyanobacterium syechocystis sp. PCC6803 (Nit6803), and an aldo‐keto reductase from Gluconobacter oxydans (Gox0644). This opens new prospects of developing computer algorithms that can effectively predict substrates for an enzyme.     

The response of stream macroinvertebrates to substrate size and heterogeneity

Manipulations of substrate size and components of heterogeneity were designed to test their independent effects and interactions on the abundance and species richness of stream macroinvertebrates. Two components of substrate heterogeneity, variation in size class proportions and number of size classes, had no independent effect on abundance or richness; and in general did not interact with median particle size. Median particle size, stream current, and detritus accounted for most of the significant variation in macroinvertebrates colonizing the experimental substrates. Rocks with high surface heterogeneity (roughness) were colonized by more individuals (but not taxa) than rocks with low surface heterogeneity.     

A vector projection method for predicting the specificity of GalNAc-transferase

The specificity of UDP-Gal-NAc:polypeptide N-acetylgalactosaminytransferase (GalNAc-transferase) is consistent with the existence of an extended site composed of nine subsites, denoted by P₄, P₃, P₂, P₁, P₀, P₁′, P₂′, P₃′, and P₄′, where the acceptor at P₀ is being either Ser or Thr. To predict whether a peptide will react with the enzyme to form a Ser- or Thr-conjugated glycopeptide, a vector projection method is proposed which uses a training set of amino acid sequences surrounding 90 Ser and 106 Thr O-glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. The model postulates independent interactions of the 9 amino acid moieties with their respective binding sites. The high ratio of correct predictions vs. total predictions for the data in both the training and the testing sets indicates that the method is self-consistent and efficient. It provides a rapid means for predicting O-glycosylation and designing effective inhibitors of GalNAc-transferase. © 1995 Wiley-Liss, Inc.     

Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity.

Cathepsin D (CatD) is a member of the mammalian aspartic protease family and is involved in cellular protein degradation and in several pathological processes. A sensitive and specific assay for the determination of CatD activity in biological samples was developed. The peptide amide substrates Amca-EDKPILF downward arrowFRLGK(biotin)-CONH2 (I), Amca-EEKPIC(Acm)F downward arrowFRLGK(biotin)-CONH2 (II) and Amca-EEKPISF downward arrowFRLGK(biotin)-CONH2 (III) contain a CatD cleavage site (F downward arrowF) flanked by a N-terminal Amca-fluorophore (7-amino-4-methylcoumarin-3-acetic acid) and a C-terminal biotin moiety. Substrates II and III proved to be specific substrates containing only one cleavage site for CatD. After cleavage of the Phe-Phe bond by CatD all biotin conjugated peptides were removed with streptavidin-coated magnetic beads. The remaining fluorescent peptides in solution represent the amount of digested substrate. The versatility of this CatD digest and pull down assay was demonstrated by measuring the activity of CatD in different subcellular fractions of human EBV-transformed B cells and human monocytes. The described method based on the designed CatD substrates represents a valuable tool for routine assays.     

Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation

Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)_n-Nop-GGK-NH₂, X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.     

Protein tyrosine phosphatase SHP-1 specifically recognizes C-terminal residues of its substrates via helix alpha0

The catalytic domain of protein tyrosine phosphatase SHP-1 possesses distinct substrate specificity. It recognizes the P-3 to P-5 residues of its substrates via the beta5-loop-beta6 region. To study the substrate specificity further, we determined the structure of the catalytic domain of SHP-1 (C455S) complexed with a less-favorable-substrate peptide originated from SIRPalpha. The complex has disordered N-terminal peptide structure and reduced interactions between the N-terminal peptide and the beta5-loop-beta6 region. This could be the basis for the lower affinity of peptide pY(427) for the catalytic domain of SHP-1. In addition, by comparing the SHP-1/less-favorable peptide complex structure with the SHP-1/substrate complex structures, we identified a novel substrate-recognition site in the catalytic domain of SHP-1. This site was formed by helix alpha0 and the alpha5-loop-alpha6 motif of SHP-1, and specifically bound residues at the P + 4 and further C-terminal positions of peptide substrates.     

小麦秸秆复合基质的理化指标及其对黄瓜幼苗生长和光合参数的影响

以小麦(Triticum aestivum L.)秸秆基质(发酵)为主,按不同比例分别添加蛭石、珍珠岩和草炭,配制10种复合基质;以不含小麦秸秆基质的复合基质为对照,对各基质的基本理化性状进行了分析和比较,并研究了不同基质对黄瓜(Cucumis sativus L.)幼苗生长、叶片叶绿素含量和光合参数的影响。结果显示:不同基质的理化指标有明显差异,小麦秸秆基质与蛭石、珍珠岩和草炭混配均降低或显著降低了基质的容重、总孔隙度、持水孔隙度、水气比、小颗粒含量、pH值和电导率,提高了通气孔隙度以及大、中颗粒的含量。按V(小麦秸秆基质)∶V(蛭石)∶V(草炭)=50∶25∶25的比例配制的复合基质T9的容重、总孔隙度、通气孔隙度、持水孔隙度、水气比和电导率分别为0.33g.cm-3、68.1%、15.3%、52.8%、3.45和3.91 mS.cm-1,理化性状最佳,具有良好的吸水和保水性能。总体上看,随复合基质中小麦秸秆基质比例的降低,黄瓜幼苗的株高、茎粗、根总长、根总体积、平均单根直径和根尖总数、生物量和壮苗指数以及叶片的叶绿素含量均呈上升趋势;复合基质中小麦秸秆基质比例较高,黄瓜叶片的净光合速率、气孔导度和蒸腾速率均较低,但与对照相比,不同基质对叶片胞间CO2浓度及水分利用效率无显著影响。在复合基质T9中黄瓜幼苗的这些生长指标、壮苗指数以及叶片叶绿素含量和光合参数总体上高于或显著高于对照和其他复合基质,生长良好。结果表明:复合基质T9较适合黄瓜幼苗的生长发育,可作为黄瓜育苗基质。     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.     

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.     

Effect of media composition on yield values of bacteria growing on binary and ternary substrate mixtures in continuous culture.

Pseudomonas aeruginosa 142 and a presumed variant were grown axenically in chemostats on salicylate/benzoate or salicylate/glucose binary feeds. Each substrate was supplied at 2, 10, 50, 90, 98, or 100% of the total energy flux. Two experiments were also run with ternary mixtures using the same substrates. Aliquots were transferred to fed-batch reactors receiving the same substrates at the same specific rates as the chemostat, but with one substrate radiolabeled with 14C. Radiolabel incorporated into biomass, 14CO2, and soluble microbial products over a period of 8 minutes was used to establish the biomass yield, CO2 yield, and product yield, respectively, associated with a given substrate. The effect of the percent substrate in the feed on the yields depended on the pair of substrates supplied. When benzoate comprised 50% or more of the applied substrate in salicylate/benzoate feeds, the fraction of benzoate in the feed had a small effect on the yield values associated with benzoate. However, when benzoate constituted 2% or 10% of the feed, CO2 yields were lower, biomass yields were slightly lower, and product yields were higher. In contrast, the percent of salicylate in the feed had little effect on any of the salicylate yields for cells growing on the salicylate/benzoate feeds. When salicylate was mixed with glucose, the yields associated with salicylate behaved quite differently. Biomass and CO2 yields were lower and product yields higher when salicylate was 2% or 10% of the feed than when it was higher. In the same substrate mixtures, glucose-based biomass yields were higher and CO2 yields were lower when glucose constituted 2% or 10% of the feed but were constant for higher percentages. The results suggest that the fate of a substrate is relatively independent of the feed composition as long as the substrate in question constitutes a significant percentage of the mixture. Thus, in those situations the assumption of a constant biomass yield in multicomponent substrate modeling is justified. However, when a given substrate constitutes a small percentage of the feed, significant changes in yield may occur.