Crystal structure of the covalent intermediate of amylosucrase from Neisseria polysaccharea

The alpha-retaining amylosucrase from the glycoside hydrolase family 13 performs a transfer reaction of a glucosyl moiety from sucrose to an acceptor molecule. Amylosucrase has previously been shown to be able to use alpha-D-glucopyranosyl fluoride as a substrate, which suggested that it could also be used for trapping the reaction intermediate for crystallographic studies. In this paper, the crystal structure of the acid/base catalyst mutant, E328Q, with a covalently bound glucopyranosyl moiety is presented. Sucrose cocrystallized crystals were soaked with alpha-D-glucopyranosyl fluoride, which resulted in the trapping of a covalent intermediate in the active site of the enzyme. The structure is refined to a resolution of 2.2 A and showed that binding of the covalent intermediate resulted in a backbone movement of 1 A around the location of the nucleophile, Asp286. This structure reveals the first covalent intermediate of an alpha-retaining glycoside hydrolase where the glucosyl moiety is identical to the expected biologically relevant entity. Comparison to other enzymes with anticipated glucosylic covalent intermediates suggests that this structure is a representative model for such intermediates. Analysis of the active site shows how oligosaccharide binding disrupts the putative nucleophilic water binding site found in the hydrolases of the GH family 13. This reveals important parts of the structural background for the shift in function from hydrolase to transglycosidase seen in amylosucrase.     

Analyzing protein functions in four dimensions

Time-resolved structural studies on biomolecular function are coming of age. Focus has shifted from studies on 'systems of opportunities' to a more problem-oriented approach, addressing significant questions in biology and chemistry. An important step in this direction has been the use of physical and chemical trapping methods to capture and then freeze reaction intermediates in crystals. Subsequent monochromatic data collection at cryogenic temperatures can produce high resolution structures of otherwise elusive intermediates. The combination of diffraction methods with spectroscopic techniques provides a means to directly correlate electronic transitions with structural transitions in the sample, eliminating much of the guesswork from experiments. Studies on cytochrome P450, isopenicillin N synthase, cytochrome cd1 nitrite reductase, copper amine oxidase and bacteriorhodopsin were selected as examples, and the results are discussed.     

Time-resolved protein crystallography.

Advances in synchrotron radiation technology have allowed exposure times from protein crystals of the order of milliseconds to be used routinely, and in exceptional circumstances exposure times of 100 ps have been obtained. However, many data sets take seconds to record because of the slow time scale of film change or crystal reorientation or translation when more than one exposure is required. This problem has been addressed by Amemiya et al. (1989). There has been considerable progress in methods to initiate reactions in protein crystals, especially the development of photolabile caged compounds but also temperature jump, pH jump, and diffusion. Although flash lamps deliver pulses of 100 mJ/ms, often several pulses are required to release sufficient product, and reaction initiation can take several seconds. Laser illumination can provide more powerful input, but the laser must be accommodated within the restricted space at the synchrotron station. The requirement to maintain synchrony among the molecules in the crystal lattice as the reaction proceeds and to ensure that the lifetime of intermediates is longer than data collection rates emphasizes the need for chemical characterization of the reaction under study. As Ringe advocated in the studies with chymotrypsin, it may be more profitable to devise conditions under which certain intermediates along the reaction pathway accumulate in the crystal and to record these in a series of discrete steps rather than continuous monitoring of the reaction. The Laue method is limited to those proteins that give well-ordered crystals and problems of transient disorder on initiation of reaction and problems of radiation damage need to be overcome or avoided by suitable experimental protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stalking intermediates in oxygen activation by iron enzymes: motivation and method

The study of high-valent-iron enzyme intermediates began in the mid-1900s with the discovery of compounds I (or ES) and II in the heme peroxidases, progressed to non-heme-diiron enzymes in the 1990s with the detection and characterization of the Fe(III)-Fe(IV) complex, X, and the Fe(IV)-Fe(IV) complex, Q, in O(2) activation by ribonucleotide reductase R2 (RNR-R2) and soluble methane monooxygenase (sMMO), respectively, and was most recently extended to mononuclear non-heme-iron oxygenases with the trapping and spectroscopic characterization of the Fe(IV)-oxo intermediate, J, in the reaction of taurine:alpha-ketoglutarate dioxygenase (TauD). Individually, each of these landmark studies helped reveal the chemical logic of that particular enzyme system. Collectively, they have significantly advanced our understanding of Nature's strategies for oxidative transformation of biomolecules (both natural and "xenobiotic"). With high-valent complexes now having been described in representatives of three major classes of iron enzymes, it is an appropriate time to ask whether and what additional insights might be gleaned from further stalking of related intermediates in other systems. In this review, we advocate that there is still much to be learned from this pursuit and summarize the insight provided by two of the landmark discoveries mentioned above (the latter two) and the subsequent studies that they spurred to support our contention. In addition, we attempt to provide, to the extent that it is possible to do so, a "how-to" guide for detection and characterization of such intermediates, focusing primarily on enzymes in which they form by activation of molecular oxygen. In this latter objective, we have drawn from an earlier review by Johnson (Enzymes, third ed. vol. 20, 1992, pp. 1-61) covering, more generally, dissection of enzyme reaction pathways by transient-state kinetic methods. That work elegantly illustrated that, although it may be impossible to develop a true algorithm for the process, a synthesis of guidelines and general principles can be of considerable value.     

Stabilization of noncovalent intermediates in enzymatically catalyzed reactions

Reactive intermediate enzyme complexes are difficult to study directly and the use of physical methods requiring observation periods of more than a second has not been possible heretofore. Here we introduce a simple approach, the "Le Chatelier forcing method" which does for the first time produce significant concentrations of such kinetically competent central intermediates observable for extended periods of time. The method involves only the forcing of the accumulation of intermediate complexes at thermodynamic equilibrium by the use of high reactant concentrations working against a high concentration of a product, combined with a valid and applicable method of analysis. We demonstrate this approach using the glutamate dehydrogenase catalyzed reaction with the reaction product ammonia as a "dam" to oppose the forward driving force of NADP and l-glutamate. We demonstrate the accumulation of substantial amounts measurable amounts of stable enzyme-NADPH-alpha-carbinolamine and alpha-iminoglutarate complexes in three different alpha-amino acid dehydrogenases. We describe the manipulation of such Le Chatelier forced equilibria to increase the prominence of particular species and discuss the implications of these findings for previously unattainable experimental approaches.     

Using species accumulation curves to estimate trapping effort in fauna surveys and species richness

Abstract The shape of species accumulation curves is influenced by the relative abundance and diversity of the fauna being sampled, and the order in which individuals are caught. We use resampling to show the variation in species accumulation curves caused by the order of trapping periods. Averaged species accumulation curves calculated by randomly assigning the order of trapping periods are smooth curves that are a better estimate of species richness and a more useful tool for determining the trapping effort required to adequately survey a site. We extend this concept of randomly resampling the trapping period to show that randomizing the number of individuals caught for each species over the number of collection periods (e.g. days) can provide an accurate estimate of the averaged species accumulation curve. This is particularly useful as it enables an accurate estimation of the proportion of the total number of species caught in an area during a survey from information on the number of individuals caught for each species and the number of trapping periods, and is not dependent on having knowledge of the trapping period in which each individual was caught. This calculation also enables an assessment to be made of the adequacy of fauna surveys to report a species inventory in environmental impact assessments when only a species list and relative abundance data are provided.     

A method for measuring the lifetime of chemically and metabolically generated electrophilic species

A method is presented for the characterization of the reaction order and rate constant for chemically and metabolically generated reactive electrophilic intermediates. The procedure employs flow kinetics and trapping of the electrophilic species. Reactive agents or metabolic intermediates are passed through a column containing a bound nucleophilic reagent. The electrophilic species reacts at a characteristic rate while moving through the column at a fixed pH, temperature and rate of flow. The alkylation products formed during this reaction are quantified in individual column segments which correspond to time intervals. This provides data for the calculation of the lifetime of the short-lived species. The method may be used for agents having half-lives of 1 s to 30 min. Metabolic intermediates need not be isolated. Data is presented for the reaction rates of 1-methyl-1-nitrosourea (MNU), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and iodomethane. A metabolic activation system is described for the conversion of acetoxymethylmethylnitrosamine (DMN-OAc) to hydroxymethylmethylnitrosamine (DMN-OH) and measurement of the stability of that intermediate. Hydroxymethylmethylnitrosamine was found to have a half-life of 28 s at pH 7.4, 37°C.     

A comparison of pitfall traps with bait traps for studying leaf litter ant communities.

A comparison of pitfall traps with bait traps for sampling leaf litter ants was studied in oak-dominated mixed forests during 1995-1997. A total of 31,732 ants were collected from pitfall traps and 54,694 ants were collected from bait traps. They belonged to four subfamilies, 17 genera, and 32 species. Bait traps caught 29 species, whereas pitfall traps caught 31 species. Bait traps attracted one species not found in pitfall traps, but missed three of the species collected with pitfall traps. Collections from the two sampling methods showed differences in species richness, relative abundance, diversity, and species accumulation curves. Pitfall traps caught significantly more ant species per plot than did bait traps. The ant species diversity obtained from pitfall traps was higher than that from bait traps. Bait traps took a much longer time to complete an estimate of species richness than did pitfall traps. Little information was added to pitfall trapping results by the bait trapping method. The results suggested that the pitfall trapping method is superior to the bait trapping method for leaf litter ant studies. Species accumulation curves showed that sampling of 2,192+/-532 ants from six plots by pitfall traps provided a good estimation of ant species richness under the conditions of this study.     

Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism.     

Mechanism of substrate recognition by drug-resistant human immunodeficiency virus type 1 protease variants revealed by a novel structural intermediate

Human immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual "intermediate" conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.     

Predator Presence and Vegetation Density Affect Capture Rates and Detectability of Litoria aurea Tadpoles: Wide-Ranging Implications for a Common Survey Technique

Trapping is a common sampling technique used to estimate fundamental population metrics of animal species such as abundance, survival and distribution. However, capture success for any trapping method can be heavily influenced by individuals’ behavioural plasticity, which in turn affects the accuracy of any population estimates derived from the data. Funnel trapping is one of the most common methods for sampling aquatic vertebrates, although, apart from fish studies, almost nothing is known about the effects of behavioural plasticity on trapping success. We used a full factorial experiment to investigate the effects that two common environmental parameters (predator presence and vegetation density) have on the trapping success of tadpoles. We estimated that the odds of tadpoles being captured in traps was 4.3 times higher when predators were absent compared to present and 2.1 times higher when vegetation density was high compared to low, using odds ratios based on fitted model means. The odds of tadpoles being detected in traps were also 2.9 times higher in predator-free environments. These results indicate that common environmental factors can trigger behavioural plasticity in tadpoles that biases trapping success. We issue a warning to researchers and surveyors that trapping biases may be commonplace when conducting surveys such as these, and urge caution in interpreting data without consideration of important environmental factors present in the study system. Left unconsidered, trapping biases in capture success have the potential to lead to incorrect interpretations of data sets, and misdirection of limited resources for managing species.     

Structural basis for antibody catalysis of a cationic cyclization reaction

Antibody 4C6 efficiently catalyzes a cationic cyclization reaction. Crystal structures of the antibody 4C6 Fab in complex with benzoic acid and in complex with its eliciting hapten were determined to 1.30A and 2.45A resolution, respectively. These crystal structures, together with computational analysis, have elucidated a possible mechanism for the monocyclization reaction. The hapten complex revealed a combining site pocket with high shape complementarity to the hapten. This active site cleft is dominated by aromatic residues that shield the highly reactive carbocation intermediates from solvent and stabilize the carbocation intermediates through cation-pi interactions. Modeling of an acyclic olefinic sulfonate ester substrate and the transition state (TS) structures shows that the chair-like transition state is favored, and trapping by water directly produces trans-2-(dimethylphenylsilyl)-cyclohexanol, whereas the less favored boat-like transition state leads to cyclohexene. The only significant change observed upon hapten binding is a side-chain rotation of Trp(L89), which reorients to form the base of the combining site. Intriguingly, a benzoic acid molecule was sequestered in the combining site of the unliganded antibody. The 4C6 active site was compared to that observed in a previously reported tandem cyclization antibody 19A4 hapten complex. These cationic cyclization antibodies exhibit convergent structural features with terpenoid cyclases that appear to be important for catalysis.     

Limited proteolysis analysis of the ribosome is affected by subunit association

Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from misfolding leading to their eventual degradation within the cell, is limited because of the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X‐ray crystallography, and cryo‐EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits. Isolated subunits were analyzed using limited proteolysis and MALDI‐MS and the results were compared with previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility on subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI‐MS can reveal structural changes to ribosomes on subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S, and 70S proteins to enable studies of ribosome assembly intermediates. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 410–422, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural and biochemical evidence for an enzymatic quinone redox cycle in Escherichia coli: identification of a novel quinol monooxygenase

Naturally synthesized quinones perform a variety of important cellular functions. Escherichia coli produce both ubiquinone and menaquinone, which are involved in electron transport. However, semiquinone intermediates produced during the one-electron reduction of these compounds, as well as through auto-oxidation of the hydroxyquinone product, generate reactive oxygen species that stress the cell. Here, we present the crystal structure of YgiN, a protein of hitherto unknown function. The three-dimensional fold of YgiN is similar to that of ActVA-Orf6 monooxygenase, which acts on hydroxyquinone substrates. YgiN shares a promoter with "modulator of drug activity B," a protein with activity similar to that of mammalian DT-diaphorase capable of reducing mendione. YgiN was able to reoxidize menadiol, the product of the "modulator of drug activity B" (MdaB) enzymatic reaction. We therefore refer to YgiN as quinol monooxygenase. Modulator of drug activity B is reported to be involved in the protection of cells from reactive oxygen species formed during single electron oxidation and reduction reactions. The enzymatic activities, together with the structural characterization of YgiN, lend evidence to the possible existence of a novel quinone redox cycle in E. coli.     

Monitoring small and arboreal mammals by camera traps: effectiveness and applications

Camera trapping has been widely applied to studies of medium to large terrestrial mammals, but its application to small arboreal mammals has hardly been tested. We employed camera trapping and other conventional monitoring methods during a mammal survey in a Site of Community Importance located within the Adda North Regional Park (Lombardy, Italy). Camera trapping was particularly successful for monitoring arboreal mammals, allowing the first detection of presence of the invasive grey squirrel (Sciurus carolinensis) in an area occupied by indigenous red squirrels (Sciurus vulgaris) and the collection of a large amount of data on squirrels and common dormice (Muscardinus avellanarius). When triggered, cameras were set to record short video clips (10 to 40 s). More than 400 events were recorded and analysed, mainly from the autumn and winter months. The daily activity pattern of both species displayed a trend from two to three activity peaks in summer to a unimodal pattern in winter, with the peaks of the two species temporally separated. Camera trapping could be a useful method also when applied to monitoring small mammals, particularly endangered arboreal or invasive alien species. For instance, the monitoring of the spread of S. carolinensis is particularly important, where the early detection of new population can be crucial for the conservation of indigenous European species. Camera trapping can be an effective addition to traditional survey methods. It provides a simple non-invasive technique for collecting a large amount of data per device with limited human effort.     

Quantitative spin trapping and triplet quenching by radicals for in vivo studies. I. The chemical model

In order to determine quantitatively the free radical content and its changes affected by additives using spin trapping under in vivo conditions, an approach is suggested carrying out experiments in a completely mixed open system (CMOS). Measurements have been carried out for a chemical oxidation process as a model system, and analysis of products and of the spin trap was extended by kinetic ESR spectrometry of the spin adducts. Since in a CMOS differential equations of accumulation of all species can be transformed into algebraic expressions using available rate constants for the formation of the spin adducts, corresponding concentrations of free radicals have been calculated. In addition, it has been established that triplet excited photosensitizers have a double effect: increasing the rate of initiation by decomposing hydroperoxide-type compounds and inhibiting the overall process by interactions with free radicals. Results indicate that by changing the "reaction vessel" the method can be applied for ex vivo and in vivo systems.     

Detecting mammals in heterogeneous landscapes: implications for biodiversity monitoring and management

With terrestrial mammals facing worldwide declines there is an increasing need to effectively monitor populations so that appropriate conservation actions can be taken. There are many techniques available to survey terrestrial mammals and in recent years there have been a number of studies comparing the effectiveness of different methods. Most of these studies have not considered complementarity (the degree to which techniques detect unique species) and effectiveness across ecological gradients. In this study we examined three widely used techniques, camera trapping, live trapping and hair detection, for their complementarity across a vegetation and disturbance gradient. Overall, camera trapping detected more species than any other single technique, but live trapping complemented the cameras by consistently detecting unique species. Additionally, technique effectiveness differed between vegetation types; cameras alone were most effective in dry forest systems while cameras combined with live traps were most effective in wetter forest systems. These results suggest that care needs to be taken when sampling across heterogeneous landscapes because relying on one technique alone could result in certain taxa being systematically overlooked, leading to potentially erroneous conclusions.     

Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis

Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 A resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.