Significance of Acquisition Parameters for Adipose Tissue Segmentation on CT Images.

OBJECTIVE. CT-based body composition analysis quantifies skeletal muscle and adipose tissue. However, acquisition parameters and quality can vary between CT images obtained for clinical care, which may lead to unreliable measurements and systematic error. The purpose of this study was to estimate the influence of IV contrast medium, tube current-exposure time product, tube potential, and slice thickness on cross-sectional area (CSA) and mean attenuation of subcutaneous (SAT), visceral (VAT), and inter-muscular adipose tissue (IMAT). MATERIALS AND METHODS. We retrospectively analyzed 244 images from 105 patients. We applied semiautomated threshold-based segmentation to CTA, dual-energy CT, and CT images acquired as part of PET examinations. An axial image at the level of the third lumbar vertebral body was extracted from each examination to generate 139 image pairs. Images from each pair were obtained with the same scanner, from the same patient, and during the same examination. Each image pair varied in only one acquisition parameter, which allowed us to estimate the effect of the parameter using one-sample t or median tests and Bland-Altman plots. RESULTS. IV contrast medium application reduced CSA in each adipose tissue compartment, with percentage change ranging from -0.4% (p = .03) to -9.3% (p < .001). Higher tube potential reduced SAT CSA (median percentage change, -4.2%; p < .001) and VAT CSA (median percentage change, -2.8%; p = .001) and increased IMAT CSA (median percentage change, -5.4%; p = .001). Thinner slices increased CSA in the VAT (mean percentage change, 3.0%; p = .005) and IMAT (median percentage change, 17.3%; p < .001) compartments. Lower tube current-exposure time product had a variable effect on CSA (median percentage change, -3.2% for SAT [p < .001], -12.6% for VAT [p = .001], and 58.8% for IMAT [p < .001]). IV contrast medium and higher tube potential increased mean attenuation, with percentage change ranging from 0.8% to 1.7% (p < .05) and from 6.2% to 20.8% (p < .001), respectively. Conversely, thinner slice and lower tube current-exposure time product reduced mean attenuation, with percentage change ranging from -5.4% to -1.0% (p < .001) and from -8.7% to -1.8% (p < .001), respectively. CONCLUSION. Acquisition parameters significantly affect CSA and mean attenuation of adipose tissue. Details of acquisition parameters used for CT-based body composition analysis need to be scrutinized and reported to facilitate interpretation of research studies.

Constructing Spaces and Times for Tactical Analysis in Football.

A possible objective in analyzing trajectories of multiple simultaneously moving objects, such as football players during a game, is to extract and understand the general patterns of coordinated movement in different classes of situations as they develop. For achieving this objective, we propose an approach that includes a combination of query techniques for flexible selection of episodes of situation development, a method for dynamic aggregation of data from selected groups of episodes, and a data structure for representing the aggregates that enables their exploration and use in further analysis. The aggregation, which is meant to abstract general movement patterns, involves construction of new time-homomorphic reference systems owing to iterative application of aggregation operators to a sequence of data selections. As similar patterns may occur at different spatial locations, we also propose constructing new spatial reference systems for aligning and matching movements irrespective of their absolute locations. The approach was tested in application to tracking data from two Bundesliga games of the 2018/2019 season. It enabled detection of interesting and meaningful general patterns of team behaviors in three classes of situations defined by football experts. The experts found the approach and the underlying concepts worth implementing in tools for football analysts.

Acoustical and Optical Determination of Mechanical Properties of Inorganically-Bound Foundry Core Materials.

Inorganically-bound sand cores are used in many light-metal foundries to form cavities in the cast part, which cannot be realised by the mould itself. To enable FEM simulations with core materials, their mechanical properties have to be measured. In this article, we adapt methods to determine the Young's and shear modulus, the Poisson ratio and the fracture strain of sand cores. This allows us to fully parametrise an ideal brittle FEM model. We found that the Young's and shear modulus can be obtained acoustically via the impulse excitation technique. The fracture strain was measured with a high-speed camera and a digital image correlation algorithm.

Quantifying the effect of slice thickness, intravenous contrast and tube current on muscle segmentation: Implications for body composition analysis.

OBJECTIVES: To quantify the effect of IV contrast, tube current and slice thickness on skeletal muscle cross-sectional area (CSA) and density (SMD) on routine CT. METHODS: CSA and SMD were computed on 216 axial CT images obtained at the L3 level in 72 patients with variations in IV contrast, slice thickness and tube current. Intra-patient mean difference (MD), 95 % CI and limits of agreement were calculated using the Bland-Altman approach. Inter- and intra-analyst agreement was evaluated. RESULTS: IV contrast significantly increased CSA by 1.88 % (MD 2.33 cm2; 95 % CI 1.76-2.89) and SMD by 5.99 % (p<0.0001). Five mm slice thickness significantly increased mean CSA by 1.11 % compared to 2 mm images (1.32 cm2; 0.78-1.85) and significantly decreased SMD by 11.64 % (p<0.0001). Low tube current significantly decreased mean CSA by 4.79 % (6.44 cm2; 3.78-9.10) and significantly increased SMD by 46.46 % (p<0.0001). Inter- and intra-analyst agreement was excellent. CONCLUSIONS: IV contrast, slice thickness and tube current significantly affect CSA and SMD. Investigators designing and analysing clinical trials using CT for body composition analysis should report CT acquisition parameters and consider the effect of slice thickness, IV contrast and tube current on myometric data. KEY POINTS: • Intravenous contrast, slice thickness and tube current significantly affect myometric data. • Image acquisition parameter variations may obscure intrapatient muscle differences on serial measurements. • Investigators using CT for body composition analysis should report CT acquisition parameters.

Sarcopenia Is Associated with Quality of Life and Depression in Patients with Advanced Cancer.

BACKGROUND: Patients with advanced cancer often experience muscle wasting (sarcopenia), yet little is known about the characteristics associated with sarcopenia and the relationship between sarcopenia and patients' quality of life (QOL) and mood. MATERIALS AND METHODS: As part of a randomized trial, we assessed baseline QOL (Functional Assessment of Cancer Therapy-General [FACT-G]) and mood (Hospital Anxiety and Depression Scale [HADS]) in patients within 8 weeks of diagnosis of incurable lung or gastrointestinal cancer, and prior to randomization. Using computed tomography scans collected as part of routine clinical care, we assessed sarcopenia at the level of the third lumbar vertebra with validated sex-specific cutoffs. We used logistic regression to explore characteristics associated with presence of sarcopenia. To examine associations between sarcopenia, QOL and mood, we used linear regression, adjusted for patients' age, sex, marital status, education, and cancer type. RESULTS: Of 237 participants (mean age = 64.41 ± 10.93 years), the majority were male (54.0%) and married (70.5%) and had lung cancer (56.5%). Over half had sarcopenia (55.3%). Older age (odds ratio [OR] = 1.05, p = .002) and education beyond high school (OR = 1.95, p = .047) were associated with greater likelihood of having sarcopenia, while female sex (OR = 0.25, p < .001) and higher body mass index (OR = 0.79, p < .001) correlated with lower likelihood of sarcopenia. Sarcopenia was associated with worse QOL (FACT-G: B = -4.26, p = .048) and greater depression symptoms (HADS-depression: B = -1.56, p = .005). CONCLUSION: Sarcopenia was highly prevalent among patients with newly diagnosed, incurable cancer. The associations of sarcopenia with worse QOL and depression symptoms highlight the need to address the issue of sarcopenia early in the course of illness. IMPLICATIONS FOR PRACTICE: This study found that sarcopenia, assessed using computed tomography scans acquired as part of routine clinical care, is highly prevalent in patients with newly diagnosed, incurable cancer. Notably, patients with sarcopenia reported worse quality of life and greater depression symptoms than those without sarcopenia. These findings highlight the importance of addressing muscle loss early in the course of illness among patients with incurable cancer. In the future, investigators should expand upon these findings to develop strategies for assessing and treating sarcopenia while striving to enhance the quality of life and mood outcomes of patients with advanced cancer.

Clustering Trajectories by Relevant Parts for Air Traffic Analysis.

Clustering of trajectories of moving objects by similarity is an important technique in movement analysis. Existing distance functions assess the similarity between trajectories based on properties of the trajectory points or segments. The properties may include the spatial positions, times, and thematic attributes. There may be a need to focus the analysis on certain parts of trajectories, i.e., points and segments that have particular properties. According to the analysis focus, the analyst may need to cluster trajectories by similarity of their relevant parts only. Throughout the analysis process, the focus may change, and different parts of trajectories may become relevant. We propose an analytical workflow in which interactive filtering tools are used to attach relevance flags to elements of trajectories, clustering is done using a distance function that ignores irrelevant elements, and the resulting clusters are summarized for further analysis. We demonstrate how this workflow can be useful for different analysis tasks in three case studies with real data from the domain of air traffic. We propose a suite of generic techniques and visualization guidelines to support movement data analysis by means of relevance-aware trajectory clustering.

Lumbar skeletal muscle index derived from routine computed tomography exams predict adverse post-extubation outcomes in critically ill patients.

PURPOSE: To evaluate the effect of a skeletal muscle index derived from a routine CT image at the level of vertebral body L3 (L3SMI) on outcomes of extubated patients in the surgical intensive care unit. MATERIALS AND METHODS: 231 patients of a prospective observational trial (NCT01967056) who had undergone CT within 5days of extubation were included. L3SMI was computed using semi-automated segmentation. Primary outcomes were pneumonia within 30days of extubation, adverse discharge disposition and 30-day mortality. Secondary outcomes included re-intubation within 72h, total hospital costs, ICU length of stay (LOS), post-extubation LOS and total hospital LOS. Outcomes were analyzed using multivariable regression models with a priori-defined covariates height, gender, age, APACHE II score and Charlson Comorbidity Index. RESULTS: L3SMI was an independent predictor of pneumonia (aOR 0.96; 95% CI 0.941-0.986; P=0.002), adverse discharge disposition (aOR 0.98; 95% CI 0.957-0.999; P=0.044) and 30-day mortality (aOR 0.94; 95% CI 0.890-0.995; P=0.033). L3SMI was significantly lower in re-intubated patients (P=0.024). Secondary analyses suggest that L3SMI is associated with total hospital costs (P=0.043) and LOS post-extubation (P=0.048). CONCLUSION: The lumbar skeletal muscle index, derived from routine abdominal CT, is an objective prognostic tool at the time of extubation.

The Arnon-Buchanan cycle: a retrospective, 1966-2016.

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO2, reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO2. Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

Pixel-Level Deep Segmentation: Artificial Intelligence Quantifies Muscle on Computed Tomography for Body Morphometric Analysis.

Pretreatment risk stratification is key for personalized medicine. While many physicians rely on an "eyeball test" to assess whether patients will tolerate major surgery or chemotherapy, "eyeballing" is inherently subjective and difficult to quantify. The concept of morphometric age derived from cross-sectional imaging has been found to correlate well with outcomes such as length of stay, morbidity, and mortality. However, the determination of the morphometric age is time intensive and requires highly trained experts. In this study, we propose a fully automated deep learning system for the segmentation of skeletal muscle cross-sectional area (CSA) on an axial computed tomography image taken at the third lumbar vertebra. We utilized a fully automated deep segmentation model derived from an extended implementation of a fully convolutional network with weight initialization of an ImageNet pre-trained model, followed by post processing to eliminate intramuscular fat for a more accurate analysis. This experiment was conducted by varying window level (WL), window width (WW), and bit resolutions in order to better understand the effects of the parameters on the model performance. Our best model, fine-tuned on 250 training images and ground truth labels, achieves 0.93 ± 0.02 Dice similarity coefficient (DSC) and 3.68 ± 2.29% difference between predicted and ground truth muscle CSA on 150 held-out test cases. Ultimately, the fully automated segmentation system can be embedded into the clinical environment to accelerate the quantification of muscle and expanded to volume analysis of 3D datasets.

Revealing Patterns and Trends of Mass Mobility Through Spatial and Temporal Abstraction of Origin-Destination Movement Data.

Origin-destination (OD) movement data describe moves or trips between spatial locations by specifying the origins, destinations, start, and end times, but not the routes travelled. For studying the spatio-temporal patterns and trends of mass mobility, individual OD moves of many people are aggregated into flows (collective moves) by time intervals. Time-variant flow data pose two difficult challenges for visualization and analysis. First, flows may connect arbitrary locations (not only neighbors), thus making a graph with numerous edge intersections, which is hard to visualize in a comprehensible way. Even a single spatial situation consisting of flows in one time step is hard to explore. The second challenge is the need to analyze long time series consisting of numerous spatial situations. We present an approach facilitating exploration of long-term flow data by means of spatial and temporal abstraction. It involves a special way of data aggregation, which allows representing spatial situations by diagram maps instead of flow maps, thus reducing the intersections and occlusions pertaining to flow maps. The aggregated data are used for clustering of time intervals by similarity of the spatial situations. Temporal and spatial displays of the clustering results facilitate the discovery of periodic patterns and longer-term trends in the mass mobility behavior.

SimpliFly: A Methodology for Simplification and Thematic Enhancement of Trajectories.

Movement data sets collected using today's advanced tracking devices consist of complex trajectories in terms of length, shape, and number of recorded positions. Multiple additional attributes characterizing the movement and its environment are often also included making the level of complexity even higher. Simplification of trajectories can improve the visibility of relevant information by reducing less relevant details while maintaining important movement patterns. We propose a systematic stepwise methodology for simplifying and thematically enhancing trajectories in order to support their visual analysis. The methodology is applied iteratively and is composed of: (a) a simplification step applied to reduce the morphological complexity of the trajectories, (b) a thematic enhancement step which aims at accentuating patterns of movement, and (c) the representation and interactive exploration of the results in order to make interpretations of the findings and further refinement to the simplification and enhancement process. We illustrate our methodology through an analysis example of two different types of tracks, aircraft and pedestrian movement.

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,ß-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

Unfamiliar metabolic links in the central carbon metabolism.

The central carbon metabolism of all organisms is considered to follow a well established fixed scheme. However, recent studies of autotrophic carbon fixation in prokaryotes revealed unfamiliar metabolic links. A new route interconnects acetyl-coenzyme A (CoA) via 3-hydroxypropionate with succinyl-CoA. Succinyl-CoA in turn may be metabolized via 4-hydroxybutyrate to two molecules of acetyl-CoA; a reversal of this route would result in the assimilation of two molecules of acetyl-CoA into C4 compounds. C5-dicarboxylic acids are a rather neglected class of metabolites; yet, they play a key role not only in one of the CO2 fixation cycles, but also in two acetate assimilation pathways that replace the glyoxylate cycle. C5 compounds such as ethylmalonate, methylsuccinate, methylmalate, mesaconate, itaconate and citramalate or their CoA esters are thereby linked to the acetyl-CoA, propionyl-CoA, glyoxylate and pyruvate pools. A novel carboxylase/reductase converts crotonyl-CoA into ethylmalonyl-CoA; similar reductive carboxylations apply to other alpha-beta-unsaturated carboxy-CoA thioesters. These unfamiliar metabolic links may provide useful tools for metabolic engineering.

Structural basis for a bispecific NADP+ and CoA binding site in an archaeal malonyl-coenzyme A reductase.

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP(+) and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP(+) and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3'- and 2'-ribose phosphate group of CoA and NADP(+), respectively, but a different one for the common ADP part: the ß-phosphate of CoA aligns with the α-phosphate of NADP(+). Evolution from an NADP(+) to a bispecific NADP(+) and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP(+) binding. Based on the paralogous aspartate-ß-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.

Molybdoenzyme that catalyzes the anaerobic hydroxylation of a tertiary carbon atom in the side chain of cholesterol.

Cholesterol is a ubiquitous hydrocarbon compound that can serve as substrate for microbial growth. This steroid and related cyclic compounds are recalcitrant due to their low solubility in water, complex ring structure, the presence of quaternary carbon atoms, and the low number of functional groups. Aerobic metabolism therefore makes use of reactive molecular oxygen as co-substrate of oxygenases to hydroxylate and cleave the sterane ring system. Consequently, anaerobic metabolism must substitute oxygenase-catalyzed steps by O(2)-independent hydroxylases. Here we show that one of the initial reactions of anaerobic cholesterol metabolism in the ß-proteobacterium Sterolibacterium denitrificans is catalyzed by an unprecedented enzyme that hydroxylates the tertiary C25 atom of the side chain without molecular oxygen forming a tertiary alcohol. This steroid C25 dehydrogenase belongs to the dimethyl sulfoxide dehydrogenase molybdoenzyme family, the closest relative being ethylbenzene dehydrogenase. It is a heterotrimer, which is probably located at the periplasmic side of the membrane and contains one molybdenum cofactor, five [Fe-S] clusters, and one heme b. The draft genome of the organism contains several genes coding for related enzymes that probably replace oxygenases in steroid metabolism.

Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use.

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO(2). Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO(2) fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

Anaerobic metabolism of indoleacetate.

The anaerobic metabolism of indoleacetate (indole-3-acetic acid [IAA]) in the denitrifying betaproteobacterium Azoarcus evansii was studied. The strain oxidized IAA completely and grew with a generation time of 10 h. Enzyme activities that transformed IAA were present in the soluble cell fraction of IAA-grown cells but were 10-fold downregulated in cells grown on 2-aminobenzoate or benzoate. The transformation of IAA did not require molecular oxygen but required electron acceptors like NAD(+) or artificial dyes. The first products identified were the enol and keto forms of 2-oxo-IAA. Later, polar products were observed, which could not yet be identified. The first steps likely consist of the anaerobic hydroxylation of the N-heterocyclic pyrrole ring to the enol form of 2-oxo-IAA, which is catalyzed by a molybdenum cofactor-containing dehydrogenase. This step is probably followed by the hydrolytic ring opening of the keto form, which is catalyzed by a hydantoinase-like enzyme. A comparison of the proteome of IAA- and benzoate-grown cells identified IAA-induced proteins. Owing to the high similarity of A. evansii with strain EbN1, whose genome is known, we identified a cluster of 14 genes that code for IAA-induced proteins involved in the early steps of IAA metabolism. These genes include a molybdenum cofactor-dependent dehydrogenase of the xanthine oxidase/aldehyde dehydrogenase family, a hydantoinase, a coenzyme A (CoA) ligase, a CoA transferase, a coenzyme B(12)-dependent mutase, an acyl-CoA dehydrogenase, a fusion protein of an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, and a periplasmic substrate binding protein for ABC transport as well as a transcriptional regulator of the GntR family. Five predicted enzymes form or act on CoA thioesters, indicating that soon after the initial oxidation of IAA and possibly ring opening, CoA thioesters are formed, and the carbon skeleton is rearranged, followed by a CoA-dependent thiolytic release of another CoA thioester. We propose a scheme of an anaerobic IAA metabolic pathway that ultimately leads to 2-aminobenzoyl-CoA or benzoyl-CoA.

An oxygenase that forms and deoxygenates toxic epoxide.

Catabolism may give rise to toxic intermediates that compromise cell vitality, such as epoxide formation in the recently elucidated and apparently universal bacterial coenzyme A (CoA)-dependent degradation of phenylacetic acid. This compound is central to the catabolism of a variety of aromatics, such as phenylalanine, lignin-related compounds or environmental contaminants. The key phenylacetyl-CoA monooxygenase (epoxidase) of the pathway, PaaABCE, is also connected to the production of various primary and secondary metabolites, as well as to the virulence of certain pathogens. However, the enzyme complex has so far not been investigated in detail. Here we characterize the bacterial multicomponent monooxygenase PaaABCE that, surprisingly, not only transforms phenylacetyl-CoA into its ring-1,2-epoxide, but also mediates the NADPH-dependent removal of the epoxide oxygen, regenerating phenylacetyl-CoA with formation of water. We provide evidence for a catalytic di-iron centre that is probably the key to the unprecedented deoxygenation of an organic compound by an oxygenase. Presumably, the bifunctionality is vital to avoid toxic intracellular epoxide levels if the subsequent catabolic steps are impeded. Our data suggest that detoxification is assisted by two thioesterases (PaaI and PaaY) forming non-reactive breakdown products. Hence, PaaABCE may harbour an intrinsic escape mechanism from its own toxic product and represents the archetype of a bifunctional oxygenase/deoxygenase. Analogous reactions may possibly be catalysed by other di-iron epoxidases.

Microbial degradation of aromatic compounds - from one strategy to four.

Aromatic compounds are both common growth substrates for microorganisms and prominent environmental pollutants. The crucial step in their degradation is overcoming the resonance energy that stabilizes the ring structure. The classical strategy for degradation comprises an attack by oxygenases that hydroxylate and finally cleave the ring with the help of activated molecular oxygen. Here, we describe three alternative strategies used by microorganisms to degrade aromatic compounds. All three of these methods involve the use of CoA thioesters and ring cleavage by hydrolysis. However, these strategies are based on different ring activation mechanisms that consist of either formation of a non-aromatic ring-epoxide under oxic conditions, or reduction of the aromatic ring under anoxic conditions using one of two completely different systems.

Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase.

Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3 Å. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg(2+) cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality.