Nicholas J. W. Rattray

New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor–cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1′ bond breakage following FMN reduction, leading to formation of the N5–C1′ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3′–C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

Taking your breath away: metabolomics breathes life in to personalized medicine

Breath-based metabolomics (breathomics) is an exciting developing area of biotechnology that centers on the capture, identification, and quantification of volatile organic compound (VOC) patterns in human breath and their utilization as tools in the diagnosis of a broad spectrum of medical problems. With the age of personalized medicines demanding rapid bespoke diagnosis and treatment, this area of molecular diagnostics is beginning to see an upsurge in biotechnological advancement. Here, we discuss recent improvements and directions in the development of breath VOC analysis and diagnosis platforms that offer the potential for disease biomarker discovery and disease prognosis.

Proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation.

Over recent decades biotechnology has made significant advances owing to the emergence of powerful biochemical and biophysical instrumentation. The development of such technologies has enabled high-throughput assessment of compounds, the implementation of recombinant DNA technology, and large-scale manufacture of monoclonal antibodies. Such innovations have ultimately resulted in the current experienced biopharmaceutical stronghold in the therapeutic market. Yet aggregate prediction and profiling remains a challenge in the formulation of biopharmaceuticals due to artifacts associated with each analytical method. We review some emerging trends and novel technologies that offer a promising potential for accurately predicting and profiling protein aggregation at various stages of biopharmaceutical product design.

Electronic cigarette exposure triggers neutrophil inflammatory responses

BackgroundThe use of electronic cigarettes (e-cigs) is increasing and there is widespread perception that e-cigs are safe. E-cigs contain harmful chemicals; more research is needed to evaluate the safety of e-cig use. Our aim was to investigate the effects of e-cigs on the inflammatory response of human neutrophils.MethodsNeutrophils were exposed to e-cig vapour extract (ECVE) and the expression of CD11b and CD66b was measured by flow cytometry and MMP-9 and CXCL8 by ELISA. We also measured the activity of neutrophil elastase (NE) and MMP-9, along with the activation of inflammatory signalling pathways. Finally we analysed the biochemical composition of ECVE by ultra-high performance liquid chromatography mass spectrometry.ResultsECVE caused an increase in the expression of CD11b and CD66b, and increased the release of MMP-9 and CXCL8. Furthermore, there was an increase in NE and MMP-9 activity and an increase in p38 MAPK activation. We also identified several harmful chemicals in ECVE, including known carcinogens.ConclusionsECVE causes a pro-inflammatory response from human neutrophils. This raises concerns over the safety of e-cig use.

Production of alkenes and novel secondary products by P450 OleTJE using novel H2O2-generating fusion protein systems

Jeotgalicoccus sp. 8456 OleTJE (CYP152L1) is a fatty acid decarboxylase cytochrome P450 that uses hydrogen peroxide (H2O2) to catalyse production of terminal alkenes, which are industrially important chemicals with biofuel applications. We report enzyme fusion systems in which Streptomyces coelicolor alditol oxidase (AldO) is linked to OleTJE. AldO oxidizes polyols (including glycerol), generating H2O2 as a coproduct and facilitating its use for efficient OleTJE‐dependent fatty acid decarboxylation. AldO activity is regulatable by polyol substrate titration, enabling control over H2O2 supply to minimize oxidative inactivation of OleTJE and prolong activity for increased alkene production. We also use these fusion systems to generate novel products from secondary turnover of 2‐OH and 3‐OH myristic acid primary products, expanding the catalytic repertoire of OleTJE.

High-throughput metabolic screening of microalgae genetic variation in response to nutrient limitation

Microalgae produce metabolites that could be useful for applications in food, biofuel or fine chemical production. The identification and development of suitable strains require analytical methods that are accurate and allow rapid screening of strains or cultivation conditions. We demonstrate the use of Fourier transform infrared (FT-IR) spectroscopy to screen mutant strains of Chlamydomonas reinhardtii. These mutants have knockdowns for one or more nutrient starvation response genes, namely PSR1, SNRK2.1 and SNRK2.2. Limitation of nutrients including nitrogen and phosphorus can induce metabolic changes in microalgae, including the accumulation of glycerolipids and starch. By performing multivariate statistical analysis of FT-IR spectra, metabolic variation between different nutrient limitation and non-stressed conditions could be differentiated. A number of mutant strains with similar genetic backgrounds could be distinguished from wild type when grown under specific nutrient limited and replete conditions, demonstrating the sensitivity of FT-IR spectroscopy to detect specific genetic traits. Changes in lipid and carbohydrate between strains and specific nutrient stress treatments were validated by other analytical methods, including liquid chromatography–mass spectrometry for lipidomics. These results demonstrate that the PSR1 gene is an important determinant of lipid and starch accumulation in response to phosphorus starvation but not nitrogen starvation. However, the SNRK2.1 and SNRK2.2 genes are not as important for determining the metabolic response to either nutrient stress. We conclude that FT-IR spectroscopy and chemometric approaches provide a robust method for microalgae screening.

Towards Improving Point-of-Care Diagnosis of Non-malaria Febrile Illness: A Metabolomics Approach

Introduction Non-malaria febrile illnesses such as bacterial bloodstream infections (BSI) are a leading cause of disease and mortality in the tropics. However, there are no reliable, simple diagnostic tests for identifying BSI or other severe non-malaria febrile illnesses. We hypothesized that different infectious agents responsible for severe febrile illness would impact on the host metabololome in different ways, and investigated the potential of plasma metabolites for diagnosis of non-malaria febrile illness. Methodology We conducted a comprehensive mass-spectrometry based metabolomics analysis of the plasma of 61 children with severe febrile illness from a malaria-endemic rural African setting. Metabolite features characteristic for non-malaria febrile illness, BSI, severe anemia and poor clinical outcome were identified by receiver operating curve analysis. Principal Findings The plasma metabolome profile of malaria and non-malaria patients revealed fundamental differences in host response, including a differential activation of the hypothalamic-pituitary-adrenal axis. A simple corticosteroid signature was a good classifier of severe malaria and non-malaria febrile patients (AUC 0.82, 95% CI: 0.70–0.93). Patients with BSI were characterized by upregulated plasma bile metabolites; a signature of two bile metabolites was estimated to have a sensitivity of 98.1% (95% CI: 80.2–100) and a specificity of 82.9% (95% CI: 54.7–99.9) to detect BSI in children younger than 5 years. This BSI signature demonstrates that host metabolites can have a superior diagnostic sensitivity compared to pathogen-detecting tests to identify infections characterized by low pathogen load such as BSI. Conclusions This study demonstrates the potential use of plasma metabolites to identify causality in children with severe febrile illness in malaria-endemic settings.

Metabolic Profiling of Geobacter sulfurreducens during Industrial Bioprocess Scale-Up.

ABSTRACT During the industrial scale-up of bioprocesses it is important to establish that the biological system has not changed significantly when moving from small laboratory-scale shake flasks or culturing bottles to an industrially relevant production level. Therefore, during upscaling of biomass production for a range of metal transformations, including the production of biogenic magnetite nanoparticles by Geobacter sulfurreducens, from 100-ml bench-scale to 5-liter fermentors, we applied Fourier transform infrared (FTIR) spectroscopy as a metabolic fingerprinting approach followed by the analysis of bacterial cell extracts by gas chromatography-mass spectrometry (GC-MS) for metabolic profiling. FTIR results clearly differentiated between the phenotypic changes associated with different growth phases as well as the two culturing conditions. Furthermore, the clustering patterns displayed by multivariate analysis were in agreement with the turbidimetric measurements, which displayed an extended lag phase for cells grown in a 5-liter bioreactor (24 h) compared to those grown in 100-ml serum bottles (6 h). GC-MS analysis of the cell extracts demonstrated an overall accumulation of fumarate during the lag phase under both culturing conditions, coinciding with the detected concentrations of oxaloacetate, pyruvate, nicotinamide, and glycerol-3-phosphate being at their lowest levels compared to other growth phases. These metabolites were overlaid onto a metabolic network of G. sulfurreducens, and taking into account the levels of these metabolites throughout the fermentation process, the limited availability of oxaloacetate and nicotinamide would seem to be the main metabolic bottleneck resulting from this scale-up process. Additional metabolite-feeding experiments were carried out to validate the above hypothesis. Nicotinamide supplementation (1 mM) did not display any significant effects on the lag phase of G. sulfurreducens cells grown in the 100-ml serum bottles. However, it significantly improved the growth behavior of cells grown in the 5-liter bioreactor by reducing the lag phase from 24 h to 6 h, while providing higher yield than in the 100-ml serum bottles.

BreathDx – molecular analysis of exhaled breath as a diagnostic test for ventilator–associated pneumonia: protocol for a European multicentre observational study

BackgroundThe diagnosis of ventilator-associated pneumonia (VAP) remains time-consuming and costly, the clinical tools lack specificity and a bedside test to exclude infection in suspected patients is unavailable. Breath contains hundreds to thousands of volatile organic compounds (VOCs) that result from host and microbial metabolism as well as the environment. The present study aims to use breath VOC analysis to develop a model that can discriminate between patients who have positive cultures and who have negative cultures with a high sensitivity.Methods/designThe Molecular Analysis of Exhaled Breath as Diagnostic Test for Ventilator-Associated Pneumonia (BreathDx) study is a multicentre observational study. Breath and bronchial lavage samples will be collected from 100 and 53 intubated and ventilated patients suspected of VAP. Breath will be analysed using Thermal Desorption – Gas Chromatography – Mass Spectrometry (TD-GC-MS). The primary endpoint is the accuracy of cross-validated prediction for positive respiratory cultures in patients that are suspected of VAP, with a sensitivity of at least 99% (high negative predictive value).DiscussionTo our knowledge, BreathDx is the first study powered to investigate whether molecular analysis of breath can be used to classify suspected VAP patients with and without positive microbiological cultures with 99% sensitivity.Trial registrationUKCRN ID number 19086, registered May 2015; as well as registration at www.trialregister.nl under the acronym ‘BreathDx’ with trial ID number NTR 6114 (retrospectively registered on 28 October 2016).