Publications by authors named "Greg Brown"

107 Publications

Machine Learning Quantitation of Cardiovascular and Cerebrovascular Disease: A Systematic Review of Clinical Applications.

Diagnostics (Basel) 2021 Mar 19;11(3). Epub 2021 Mar 19.

Cancer Research Institute, University of South Australia, Adelaide, SA 5001, Australia.

Research into machine learning (ML) for clinical vascular analysis, such as those useful for stroke and coronary artery disease, varies greatly between imaging modalities and vascular regions. Limited accessibility to large diverse patient imaging datasets, as well as a lack of transparency in specific methods, are obstacles to further development. This paper reviews the current status of quantitative vascular ML, identifying advantages and disadvantages common to all imaging modalities. Literature from the past 8 years was systematically collected from MEDLINE and Scopus database searches in January 2021. Papers satisfying all search criteria, including a minimum of 50 patients, were further analysed and extracted of relevant data, for a total of 47 publications. Current ML image segmentation, disease risk prediction, and pathology quantitation methods have shown sensitivities and specificities over 70%, compared to expert manual analysis or invasive quantitation. Despite this, inconsistencies in methodology and the reporting of results have prevented inter-model comparison, impeding the identification of approaches with the greatest potential. The clinical potential of this technology has been well demonstrated in Computed Tomography of coronary artery disease, but remains practically limited in other modalities and body regions, particularly due to a lack of routine invasive reference measurements and patient datasets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/diagnostics11030551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003459PMC
March 2021

The moderating role of the built environment in prenatal lifestyle interventions.

Int J Obes (Lond) 2021 Feb 26. Epub 2021 Feb 26.

Department of Natural Resources Management and Environmental Sciences, Cal Poly, San Luis Obispo, CA, USA.

This study examined whether the neighborhood built environment moderated gestational weight gain (GWG) in LIFE-Moms clinical trials. Participants were 790 pregnant women (13.9 weeks' gestation) with overweight or obesity randomized within four clinical centers to standard care or lifestyle intervention to reduce GWG. Geographic information system (GIS) was used to map the neighborhood built environment. The intervention relative to standard care significantly reduced GWG (coefficient = 0.05; p = 0.005) and this effect remained significant (p < 0.03) after adjusting for built environment variables. An interaction was observed for presence of fast food restaurants (coefficient = -0.007; p = 0.003). Post hoc tests based on a median split showed that the intervention relative to standard care reduced GWG in participants living in neighborhoods with lower fast food density 0.08 [95% CI, 0.03,0.12] kg/week (p = 0.001) but not in those living in areas with higher fast food density (0.02 [-0.04, 0.08] kg/week; p = 0.55). Interaction effects suggested less intervention efficacy among women living in neighborhoods with more grocery/convenience stores (coefficient = -0.005; p = 0.0001), more walkability (coefficient -0.012; p = 0.007) and less crime (coefficient = 0.001; p = 0.007), but post-hoc tests were not significant. No intervention x environment interaction effects were observed for total number of eating establishments or tree canopy. Lifestyle interventions during pregnancy were effective across diverse physical environments. Living in environments with easy access to fast food restaurants may limit efficacy of prenatal lifestyle interventions, but future research is needed to replicate these findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41366-021-00782-wDOI Listing
February 2021

The moderating role of the built environment in prenatal lifestyle interventions.

Int J Obes (Lond) 2021 Feb 26. Epub 2021 Feb 26.

Department of Natural Resources Management and Environmental Sciences, Cal Poly, San Luis Obispo, CA, USA.

This study examined whether the neighborhood built environment moderated gestational weight gain (GWG) in LIFE-Moms clinical trials. Participants were 790 pregnant women (13.9 weeks' gestation) with overweight or obesity randomized within four clinical centers to standard care or lifestyle intervention to reduce GWG. Geographic information system (GIS) was used to map the neighborhood built environment. The intervention relative to standard care significantly reduced GWG (coefficient = 0.05; p = 0.005) and this effect remained significant (p < 0.03) after adjusting for built environment variables. An interaction was observed for presence of fast food restaurants (coefficient = -0.007; p = 0.003). Post hoc tests based on a median split showed that the intervention relative to standard care reduced GWG in participants living in neighborhoods with lower fast food density 0.08 [95% CI, 0.03,0.12] kg/week (p = 0.001) but not in those living in areas with higher fast food density (0.02 [-0.04, 0.08] kg/week; p = 0.55). Interaction effects suggested less intervention efficacy among women living in neighborhoods with more grocery/convenience stores (coefficient = -0.005; p = 0.0001), more walkability (coefficient -0.012; p = 0.007) and less crime (coefficient = 0.001; p = 0.007), but post-hoc tests were not significant. No intervention x environment interaction effects were observed for total number of eating establishments or tree canopy. Lifestyle interventions during pregnancy were effective across diverse physical environments. Living in environments with easy access to fast food restaurants may limit efficacy of prenatal lifestyle interventions, but future research is needed to replicate these findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41366-021-00782-wDOI Listing
February 2021

Cane toads (Rhinella marina) rely on water access, not drought tolerance, to invade xeric Australian environments.

Oecologia 2019 Feb 8;189(2):307-316. Epub 2018 Dec 8.

School of Life Sciences, Arizona State University, Tempe, AZ, 85287-4501, USA.

The invasion of habitats with novel environmental challenges may require physiological tolerances not seen in conspecifics from the native range. We used a combination of field and laboratory-based experiments to assess physiological tolerance to limited water access at four sites distributed across the historical invasion path of cane toads (Rhinella marina) in Australia that, from east to west, alternated between mesic and seasonally xeric habitats. Toads from all locations were well hydrated at the time of capture. However, experimental dehydration caused greater mass loss, higher plasma osmolality, and inhibition of lytic ability in toads from xeric compared to mesic locations. These results suggest somewhat surprisingly that toads from xeric environments are physiologically more vulnerable to water loss. In contrast, bactericidal ability was not sensitive to hydric state and was greater in toads from eastern (long-colonized) areas. Similar patterns in lytic ability in hydrated toads and agglutination ability in wild toads suggest that toads along the invasion front face a tradeoff between enhanced dispersal ability and physiological responses to dehydration. The ability of this invasive species to spread into drier environments may be underpinned by a combination of phenotypic plasticity and evolved (heritable) traits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00442-018-4321-1DOI Listing
February 2019

Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.

Environ Sci Technol 2018 11 15;52(21):12388-12401. Epub 2018 Oct 15.

Department of Chemical Engineering and Applied Chemistry , University of Toronto , Toronto , ON M5S 3E5 , Canada.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 Å resolution and revealed a modified α/β hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.est.8b04252DOI Listing
November 2018

Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase.

ACS Chem Biol 2018 10 4;13(10):3011-3020. Epub 2018 Oct 4.

Lewis Sigler Institute for Integrative Genomics , Princeton University , Princeton , New Jersey 08544 , United States.

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.8b00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466636PMC
October 2018

Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase.

ACS Chem Biol 2018 10 4;13(10):3011-3020. Epub 2018 Oct 4.

Lewis Sigler Institute for Integrative Genomics , Princeton University , Princeton , New Jersey 08544 , United States.

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.8b00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466636PMC
October 2018

Biosynthesis and Activity of Prenylated FMN Cofactors.

Cell Chem Biol 2018 05 15;25(5):560-570.e6. Epub 2018 Mar 15.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada. Electronic address:

Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis, biological decomposition of lignin, and biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the purification and characterization of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) can be activated by free prFMN or C2'-hydroxylated prFMN under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2018.02.007DOI Listing
May 2018

Determinants and Prediction of Esterase Substrate Promiscuity Patterns.

ACS Chem Biol 2018 01 20;13(1):225-234. Epub 2017 Dec 20.

Institute of Catalysis, Consejo Superior de Investigaciones Científicas , 28049 Madrid, Spain.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.7b00996DOI Listing
January 2018

Exploring Bacterial Carboxylate Reductases for the Reduction of Bifunctional Carboxylic Acids.

Biotechnol J 2017 Nov 5;12(11). Epub 2017 Sep 5.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, ON, M5S 3E5, Canada.

Carboxylic acid reductases (CARs) selectively reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Although CARs attracts significant interest, only a few enzymes have been characterized to date, whereas the vast majority of CARs have yet to be examined. Herein the authors report that 12 bacterial CARs reduces a broad range of bifunctional carboxylic acids containing oxo-, hydroxy-, amino-, or second carboxyl groups with several enzymes showing activity toward 4-hydroxybutanoic (4-HB) and adipic acids. These CARs exhibits significant reductase activity against substrates whose second functional group is separated from the carboxylate by at least three carbons with both carboxylate groups being reduced in dicarboxylic acids. Purified CARs supplemented with cofactor regenerating systems (for ATP and NADPH), an inorganic pyrophosphatase, and an aldo-keto reductase catalyzes a high conversion (50-76%) of 4-HB to 1,4-butanediol (1,4-BDO) and adipic acid to 1,6-hexanediol (1,6-HDO). Likewise, Escherichia coli strains expressing eight different CARs efficiently reduces 4-HB to 1,4-BDO with 50-95% conversion, whereas adipic acid is reduced to a mixture of 6-hydroxyhexanoic acid (6-HHA) and 1,6-HDO. Thus, our results illustrate the broad biochemical diversity of bacterial CARs and their compatibility with other enzymes for applications in biocatalysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/biot.201600751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681412PMC
November 2017

Identifying conflict potential in a coastal and marine environment using participatory mapping.

J Environ Manage 2017 Jul 24;197:706-718. Epub 2017 Apr 24.

Environmental and Conservation Sciences, School of Veterinary and Life Sciences, Murdoch University, South Street, Murdoch, WA, 6150, Australia. Electronic address:

Planning for coastal and marine environments is often characterized by conflict over current and proposed uses. Marine spatial planning has been proposed as a way forward, however, social data are often missing impeding decision-making. Participatory mapping, a technique useful for providing social data and predict conflict potential, is being used in an increasing number of terrestrial applications to inform planning, but has been little used in the marine realm. This study collected social data for an extensive coastline in northwestern Australia via 167 in-depth face-to-face interviews including participant mapping of place values. From the transcribed interviews and digitized maps, we inductively identified 17 values, with biodiversity, the physical landscape, and Aboriginal culture being most valued. To spatially identify conflict potential, values were classified in matrices as consumptive or non-consumptive with the former assumed to be less compatible with other values. Pairwise comparisons of value compatibilities informed a spatial GIS determination of conflict potential. The results were overlaid with the boundaries of nine marine protected areas in the region to illustrate the application of this method for marine spatial planning. The three near shore marine protected areas had at least one third of their area exhibiting conflict potential. Participatory mapping accompanied by conflict potential mapping provides important insights for spatial planning in these often-highly contested marine environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jenvman.2016.12.026DOI Listing
July 2017

Understanding the effects of different social data on selecting priority conservation areas.

Conserv Biol 2017 12 31;31(6):1439-1449. Epub 2017 Jul 31.

School of Earth and Environmental Sciences, University of Queensland, Brisbane, QLD, 4072, Australia.

Conservation success is contingent on assessing social and environmental factors so that cost-effective implementation of strategies and actions can be placed in a broad social-ecological context. Until now, the focus has been on how to include spatially explicit social data in conservation planning, whereas the value of different kinds of social data has received limited attention. In a regional systematic conservation planning case study in Australia, we examined the spatial concurrence of a range of spatially explicit social values and land-use preferences collected using a public participation geographic information system and biological data. We used Zonation to integrate the social data with the biological data in a series of spatial-prioritization scenarios to determine the effect of the different types of social data on spatial prioritization compared with biological data alone. The type of social data (i.e., conservation opportunities or constraints) significantly affected spatial prioritization outcomes. The integration of social values and land-use preferences under different scenarios was highly variable and generated spatial prioritizations 1.2-51% different from those based on biological data alone. The inclusion of conservation-compatible values and preferences added relatively few new areas to conservation priorities, whereas including noncompatible economic values and development preferences as costs significantly changed conservation priority areas (48.2% and 47.4%, respectively). Based on our results, a multifaceted conservation prioritization approach that combines spatially explicit social data with biological data can help conservation planners identify the type of social data to collect for more effective and feasible conservation actions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cobi.12947DOI Listing
December 2017

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.

Sci Rep 2017 03 8;7:44103. Epub 2017 Mar 8.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep44103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341072PMC
March 2017

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies.

Appl Environ Microbiol 2017 04 17;83(7). Epub 2017 Mar 17.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from showed the highest activity and was selected for comparative studies with STM2406 from serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in / Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates. In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.03172-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359500PMC
April 2017

Structural and functional characterization of the TYW3/Taw3 class of SAM-dependent methyltransferases.

RNA 2017 03 8;23(3):346-354. Epub 2016 Dec 8.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.

-adenosylmethionine (SAM)-dependent methyltransferases regulate a wide range of biological processes through the modification of proteins, nucleic acids, polysaccharides, as well as various metabolites. TYW3/Taw3 is a SAM-dependent methyltransferase responsible for the formation of a tRNA modification known as wybutosine and its derivatives that are required for accurate decoding in protein synthesis. Here, we report the crystal structure of Taw3, a homolog of TYW3 from , which revealed a novel α/β fold. The sequence motif (S/T)xSSCxGR and invariant aspartate and histidine, conserved in TYW3/Taw3, cluster to form the catalytic center. These structural and sequence features indicate that TYW3/Taw3 proteins constitute a distinct class of SAM-dependent methyltransferases. Using site-directed mutagenesis along with in vivo complementation assays combined with mass spectrometry as well as ligand docking and cofactor binding assays, we have identified the active site of TYW3 and residues essential for cofactor binding and methyltransferase activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.057943.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311493PMC
March 2017

Mortality reduction in patients treated with long-term intensive lipid therapy: 25-year follow-up of the Familial Atherosclerosis Treatment Study-Observational Study.

J Clin Lipidol 2016 Sep-Oct;10(5):1091-7. Epub 2016 Jul 9.

Division of Cardiology, University of Washington School of Medicine, Seattle, WA, USA.

Background: Cardiovascular disease (CVD) begins early in life and is associated with both the number of risk factors present and length of exposure to these risk factors including hyperlipidemia.

Objectives: The clinical benefit of intensive lipid therapy over 25 years was investigated in the Familial Atherosclerosis Treatment Study-Observational Study.

Methods: Of 175 coronary artery disease subjects with mean low-density lipoprotein cholesterol (LDL-C) of 191 mg/dL and mean age of 50 years, who completed the randomized and placebo-controlled Familial Atherosclerosis Treatment Study, 100 chose receiving lipid management by their physicians (usual care [UC]) and 75 elected to receive an intensive treatment [IT] for lipid management with lovastatin (40 mg/d), niacin (2.5 g/d), and colestipol (20 g/d) from 1989 to 2004, followed by double therapy with simvastatin (40-80 mg/d) and niacin from 2005 to 2006 and by triple therapy of ezetimibe 10 mg and simvastatin 40 to 80 mg/d plus niacin during 2007 to 2012. Deaths from CVD, non-CVD, and any cause were compared between UC and IT using Cox proportional hazards model.

Results: UC and IT groups were similar in risk factors with the exception that IT had more severe coronary artery disease. Mean LDL-C levels were 167 mg/dL from 1988 to 2004, 97 from 2005 to 2006, and 96 from 2007 to 2012 in surviving subjects receiving UC. IT lowered LDL-C to 119, 97, and 83 mg/dL in the 3 periods, respectively. Compared with UC, IT significantly reduced total mortality (11.1 vs 26.3 per 1000 person years [PY], hazard ratio [HR] = 0.45, 95% confidence interval [CI]: 0.26-0.77, P = .003) and CVD mortality (10.6 vs 27.7 per 1000 PY, HR = 0.34, 95% CI: 0.15-0.80, P = .009). The non-CVD mortality was also reduced but was not of statistical significance (6.8 vs 12.7 per 1000 PY, HR = 0.55, 95% CI: 0.27-1.14, P = .11).

Conclusions: Long-term intensive lipid therapy significantly reduced total and cardiovascular mortality in Familial Atherosclerosis Treatment Study-Observational Study. These results support the importance of lifetime risk management to improve long-term outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055060PMC
http://dx.doi.org/10.1016/j.jacl.2016.06.013DOI Listing
October 2017

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

Plant Cell 2016 10 27;28(10):2683-2696. Epub 2016 Sep 27.

Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1105/tpc.16.00600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134987PMC
October 2016

A system dynamics simulation model for sustainable water resources management and agricultural development in the Volta River Basin, Ghana.

Sci Total Environ 2016 Dec 27;573:444-457. Epub 2016 Aug 27.

School of Geography, Planning and Environmental Management, University of Queensland, Brisbane, QLD, 4072, Australia.

In a rapidly changing water resources system, dynamic models based on the notion of systems thinking can serve as useful analytical tools for scientists and policy-makers to study changes in key system variables over time. In this paper, an integrated system dynamics simulation model was developed using a system dynamics modelling approach to examine the feedback processes and interaction between the population, the water resource, and the agricultural production sub-sectors of the Volta River Basin in West Africa. The objective of the model is to provide a learning tool for policy-makers to improve their understanding of the long-term dynamic behaviour of the basin, and as a decision support tool for exploring plausible policy scenarios necessary for sustainable water resource management and agricultural development. Structural and behavioural pattern tests, and statistical test were used to evaluate and validate the performance of the model. The results showed that the simulated outputs agreed well with the observed reality of the system. A sensitivity analysis also indicated that the model is reliable and robust to uncertainties in the major parameters. Results of the business as usual scenario showed that total population, agricultural, domestic, and industrial water demands will continue to increase over the simulated period. Besides business as usual, three additional policy scenarios were simulated to assess their impact on water demands, crop yield, and net-farm income. These were the development of the water infrastructure (scenario 1), cropland expansion (scenario 2) and dry conditions (scenario 3). The results showed that scenario 1 would provide the maximum benefit to people living in the basin. Overall, the model results could help inform planning and investment decisions within the basin to enhance food security, livelihoods development, socio-economic growth, and sustainable management of natural resources.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scitotenv.2016.08.081DOI Listing
December 2016

Cardiac iron load and function in transfused patients treated with deferasirox (the MILE study).

Eur J Haematol 2017 Feb 20;98(2):97-105. Epub 2016 Sep 20.

Thalassaemia Services Victoria, Monash Medical Centre, Melbourne, VIC, Australia.

Objectives: To assess the effect of iron chelation therapy with deferasirox on cardiac iron and function in patients with transfusion-dependent thalassemia major, sickle cell disease (SCD), and myelodysplastic syndromes (MDS).

Methods: This phase IV, single-arm, open-label study over 53 wk evaluated the change in cardiac and liver iron load with deferasirox (up to 40 mg/kg/d), measured by magnetic resonance imaging (MRI).

Results: Cardiac iron load (myocardial T2*) significantly improved (P = 0.002) overall (n = 46; n = 36 thalassemia major, n = 4 SCD, n = 6 MDS). Results were significant for patients with normal and moderate baseline cardiac iron (P = 0.017 and P = 0.015, respectively), but not in the five patients with severe cardiac iron load. Liver iron concentration (LIC) significantly decreased overall [mean LIC 10.4 to 8.2 mg Fe/g dry tissue (dw); P = 0.024], particularly in those with baseline LIC >7 mg Fe/g dw (19.9 to 15.6 mg Fe/g dw; P = 0.002). Furthermore, myocardial T2* significantly increased in patients with LIC <7 mg Fe/g dw, but not in those with a higher LIC. Safety was consistent with previous reports.

Conclusions: Once-daily deferasirox over 1 yr significantly increased myocardial T2* and reduced LIC. This confirms that single-agent deferasirox is effective in the management of cardiac iron, especially for patients with myocardial T2* >10 ms (Clinicaltrials.gov identifier: NCT00673608).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ejh.12793DOI Listing
February 2017

Emergency transport of stroke suspects in a rural state: opportunities for improvement.

Am J Emerg Med 2016 Aug 14;34(8):1640-4. Epub 2016 Jun 14.

Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR 72205. Electronic address:

Introduction: Time delay is the key obstacle for receiving successful stroke treatment. Alteplase therapy must start within 4.5 hours from stroke occurrence. Rapid transport to a primary stroke center (PSC) or acute stroke-ready hospital (ASRH) by the emergency medical system (EMS) paramedics is vital. We determined transport time and destination data for EMS-identified and -delivered stroke suspects in Arkansas during 2013. Our objective was to analyze transport time and the hospital qualification for stroke care across the state.

Methods: The state's 75 counties were placed into 8 geographical regions (R1-R8). Transport time and hospital qualification were determined for all EMS-identified strokes. Each hospital's stroke care status was categorized as PSC, ASRH, a nonspecialty or unknown care facility (NSCF), out-of-state, or nonapplicable designation facilities.

Results: There were 9588 EMS stroke ground transports with median within-region transport times of 29-40 minutes. Statewide, only 65% of EMS-transported stroke patients were transported to either PSC (12%) or ASRH (53%) facilities. One-third of the patients (30.6%) were delivered to NSCFs, where acute stroke therapy may rarely be performed. Regions with the highest suspected-stroke cases per capita also had the highest percentage of transports to NSCFs.

Conclusion: With only a few PSCs in Arkansas, EMS agencies should prioritize transporting stroke patients to ASRHs when PSCs are not regionally located.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajem.2016.06.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4998736PMC
August 2016

A family of metal-dependent phosphatases implicated in metabolite damage-control.

Nat Chem Biol 2016 08 20;12(8):621-7. Epub 2016 Jun 20.

Horticultural Sciences Department, University of Florida, Gainesville, Florida, USA.

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg(2+) bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001580PMC
August 2016

Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

Biomacromolecules 2016 06 2;17(6):2027-39. Epub 2016 May 2.

Department of Chemical Engineering and Applied Chemistry, University of Toronto , Toronto, Ontario M5S 3E5, Canada.

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biomac.6b00223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886529PMC
June 2016

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

J Biol Chem 2015 Jul 12;290(30):18678-98. Epub 2015 Jun 12.

the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada,

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.657916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513125PMC
July 2015

Cycle of coercion: experiences of maltreatment and disciplinary measures in Canadian inmates.

Int J Prison Health 2014 ;10(2):79-93

Assistant Professor, based at Department of Criminology, University of Ottawa, Ottawa, Canada.

Purpose: In spite of past and current efforts at implementing effective rehabilitative interventions in carceral settings, institutions of confinement are primarily concerned with the maintenance of order within their walls. The purpose of this paper is to better understand associations between inmates' developmental background and the experience of institutional discipline, to collect information on childhood maltreatment and disciplinary measures for a sample of Canadian prisoners.

Design/methodology/approach: Information relative to socio-economic background, childhood maltreatment and experience of discipline while in custody was obtained using face-to-face interviews and institutional file review for a sample of 416 male and 106 female offenders in Canadian provincial institutions.

Findings: Results from logistic regression analyses provided support for the association between childhood maltreatment and the experience of discipline, specifically in the form of increased monitoring from correctional staff. Furthermore, this association was found to be more pronounced for female offenders.

Research Limitations/implications: The findings highlight the need to incorporate a developmental perspective to current understanding of the use of disciplinary interventions in a prison environment. Such an approach may allow for preventing the enactment of a cycle of coercion, with negative consequences for the inmates.

Originality/value: This study is original in its use of latent variable analytic methods to uncover the structure underlying the construct of childhood maltreatment in adult offenders. In addition, it provides valuable data of interest to researchers, corrections personnel and policy makers on the possible links between earlier developmental experiences and adjustment to the prison environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1108/IJPH-09-2013-0043DOI Listing
November 2015

A Comparison of the Efficacy and Adverse Effects of Double-Lumen Endobronchial Tubes and Bronchial Blockers in Thoracic Surgery: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

J Cardiothorac Vasc Anesth 2015 Aug 2;29(4):955-66. Epub 2014 Dec 2.

College of Medicine and Veterinary Medicine, Academic Administration, The University of Edinburgh, Edinburgh, United Kingdom.

Objective: To compare the efficacy and adverse effects of using bronchial blockers (BBs) and double-lumen endobronchial tubes (DLTs).

Design: Systematic review and meta-analysis of randomized controlled trials (RCTs) comparing BBs and DLTs.

Setting: Hospital units undertaking thoracic surgery

Participants: Patients undergoing thoracic surgery requiring lung isolation.

Interventions: BBs and DLTs.

Measurements And Main Results: A systematic literature search was conducted for RCTs comparing BBs and DLTs using Google Scholar, Ovid Medline, and Cochrane library databases up to October 2013. Inclusion criteria were RCTs comparing BBs and DLTs, intubation carried out by qualified anesthesiologists or trainee specialists, outcome measures relating to either efficacy or adverse effects. Studies that were inaccessible in English were excluded. Mantel-Haenszel fixed-effect meta-analysis of recurring outcome measures was performed using RevMan 5 software. The search produced 39 RCTs published between 1996 and 2013. DLTs were quicker to place (mean difference: 51 seconds, 95% confidence intervals [CI] 8-94 seconds; p = 0.02) and less likely to be incorrectly positioned (odds ratio [OR] 2.70; 95% CI 1.18-6.18, p = 0.02) than BBs. BBs were associated with fewer patients having a postoperative sore throat (OR 0.39, 95% CI: 0.23-0.68, p = 0.0009), less hoarseness (OR: 0.43,95%, CI 0.24-0.75, p = 0.003), and fewer airway injuries (OR 0.40, 95% CI 0.21-0.75, p = 0.005) than DLTs.

Conclusion: While BBs are associated with a lower incidence of airway injury and a lower severity of injury, DLTs can be placed quicker and more reliably.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.jvca.2014.11.017DOI Listing
August 2015

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.

Nucleic Acids Res 2015 Jan 8;43(1):530-43. Epub 2014 Dec 8.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gku1285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288178PMC
January 2015

Prolonged combination lipid therapy is associated with reduced carotid intima-media thickness: a case-control study of the 20-year Familial Atherosclerosis Treatment - Observational Study (FATS-OS).

J Clin Lipidol 2014 Sep-Oct;8(5):489-93. Epub 2014 Jul 12.

Cardiovascular Atherosclerosis Research Laboratory, Division of Cardiology, Department of Medicine, University of Washington, Seattle, WA, USA.

Background: Studies have documented the short-term vascular benefits of combination lipid therapy.

Objective: Our objective was to evaluate the long-term effects of combination lipid therapy on carotid intima-media thickness (CIMT) in patients with coronary artery disease.

Methods: We performed a case-control study in patients who had finished the Familial Atherosclerosis Treatment Study (FATS) and returned to usual care with statin therapy alone or had elected to participate in the 20-year FATS-Observational Study (FATS-OS) and received combination therapy with lovastatin (40 mg/day), niacin (2-3 g/day), and colestipol (20 gm/day) for 11 years, then continued with simvastatin (10-80 mg/day) or lovastatin (40-80 mg/day) plus niacin (2-4 g/day). After 17.8 ± 0.8 years with combination therapy and 19.0 ± 0.8 years with usual care, cholesterol levels and CIMT were collected in 43 FATS-OS patients and 26 usual care patients.

Results: Combination therapy group had a greater decrease in total cholesterol (-42 ± 14% vs -31 ± 17%, P = .008) and low-density lipoprotein cholesterol (LDL-C) (-57 ± 13% vs -38 ± 25%, P < .001) and greater increase in high-density lipoprotein cholesterol (HDL-C) (38 ± 43% vs 15 ± 23%, P = .02) as compared with usual care. CIMT (0.902 ± 0.164 vs 1.056 ± 0.169 mm, P < .001) on intensive therapy was significantly less compared with usual care. Multivariate regression analysis (coefficient, 95% CI) showed that combination therapy (-0.13; -0.21 to -0.04, P = .003) and on-therapy LDL-C (0.15; 0.02 to 0.28, P = .03) were significant independent predictors of CIMT.

Conclusions: Prolonged combination lipid therapy is associated with greater improvements in LDL-C and HDL-C levels and less atherosclerotic burden as compared with statin therapy alone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacl.2014.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4171688PMC
February 2015

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.

Nucleic Acids Res 2014 8;42(17):11144-55. Epub 2014 Sep 8.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gku797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176176PMC
February 2015

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.

Appl Microbiol Biotechnol 2015 Mar 7;99(5):2165-78. Epub 2014 Sep 7.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-014-6038-3DOI Listing
March 2015

Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus.

Appl Environ Microbiol 2014 Jul 18;80(13):3992-4002. Epub 2014 Apr 18.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.00215-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054205PMC
July 2014