Publications by authors named "Brian Fox"

180 Publications

Multifunctional cellulases are potent, versatile tools for a renewable bioeconomy.

Curr Opin Biotechnol 2021 02 4;67:141-148. Epub 2021 Feb 4.

Dept. of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI, 53706, United States; Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, 1552 University Ave, Madison, WI, 53726, United States. Electronic address:

Enzyme performance is critical to the future bioeconomy based on renewable plant materials. Plant biomass can be efficiently hydrolyzed by multifunctional cellulases (MFCs) into sugars suitable for conversion into fuels and chemicals, and MFCs fall into three functional categories. Recent work revealed MFCs with broad substrate specificity, dual exo-activity/endo-activity on cellulose, and intramolecular synergy, among other novel characteristics. Binding modules and accessory catalytic domains amplify MFC and xylanase activity in a wide variety of ways, and processive endoglucanases achieve autosynergy on cellulose. Multidomain MFCs from Caldicellulosiruptor are heat-tolerant, adaptable to variable cellulose crystallinity, and may provide interchangeable scaffolds for recombinant design. Further studies of MFC properties and their reactivity with plant biomass are recommended for increasing biorefinery yields.
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http://dx.doi.org/10.1016/j.copbio.2020.12.020DOI Listing
February 2021

Lung Organoids And Other Preclinical Models Of Pulmonary Fibrosis.

QJM 2021 Jan 23. Epub 2021 Jan 23.

Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive fatal disease affecting over 100,000 people in Europe with an increasing incidence. Available treatments offer only slowing of disease progression and are poorly tolerated by patients leading to cessation of therapy. Lung transplant remains the only cure. Therefore, alternative treatments are urgently required. The pathology of IPF is complex and poorly understood and thus creates a major obstacle to the discovery of novel treatments. Additionally, preclinical assessment of new treatments currently relies upon animal models where disparities with human lung biology often hamper drug development. At a cellular level, IPF is characterised by persistent and abnormal deposition of extracellular matrix (ECM) by fibroblasts and alveolar epithelial cell injury which is seen as a key event in initiation of disease progression. In depth investigation of the role of alveolar epithelial cells in health and disease has been impeded due to difficulties in primary cell isolation and culture ex vivo. Novel strategies employing patient derived induced pluripotent stem cells (iPSC) engineered to produce type 2 alveolar epithelial cells (iAEC2) cultured as 3 D organoids have the potential to overcome these hurdles and inform new effective precision treatments for IPF leading to improved survival and quality of life for patients worldwide.
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http://dx.doi.org/10.1093/qjmed/hcaa281DOI Listing
January 2021

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis.

J Biol Chem 2020 12;295(51):17752-17769

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA; Great Lakes Bioenergy Research Center, Madison, Wisconsin, USA. Electronic address:

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.
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http://dx.doi.org/10.1074/jbc.RA120.015328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762963PMC
December 2020

Bone marrow microenvironments that contribute to patient outcomes in newly diagnosed multiple myeloma: A cohort study of patients in the Total Therapy clinical trials.

PLoS Med 2020 11 4;17(11):e1003323. Epub 2020 Nov 4.

Myeloma Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

Background: The tumor microenvironment (TME) is increasingly appreciated as an important determinant of cancer outcome, including in multiple myeloma (MM). However, most myeloma microenvironment studies have been based on bone marrow (BM) aspirates, which often do not fully reflect the cellular content of BM tissue itself. To address this limitation in myeloma research, we systematically characterized the whole bone marrow (WBM) microenvironment during premalignant, baseline, on treatment, and post-treatment phases.

Methods And Findings: Between 2004 and 2019, 998 BM samples were taken from 436 patients with newly diagnosed MM (NDMM) at the University of Arkansas for Medical Sciences in Little Rock, Arkansas, United States of America. These patients were 61% male and 39% female, 89% White, 8% Black, and 3% other/refused, with a mean age of 58 years. Using WBM and matched cluster of differentiation (CD)138-selected tumor gene expression to control for tumor burden, we identified a subgroup of patients with an adverse TME associated with 17 fewer months of progression-free survival (PFS) (95% confidence interval [CI] 5-29, 49-69 versus 70-82 months, χ2 p = 0.001) and 15 fewer months of overall survival (OS; 95% CI -1 to 31, 92-120 versus 113-129 months, χ2 p = 0.036). Using immunohistochemistry-validated computational tools that identify distinct cell types from bulk gene expression, we showed that the adverse outcome was correlated with elevated CD8+ T cell and reduced granulocytic cell proportions. This microenvironment develops during the progression of premalignant to malignant disease and becomes less prevalent after therapy, in which it is associated with improved outcomes. In patients with quantified International Staging System (ISS) stage and 70-gene Prognostic Risk Score (GEP-70) scores, taking the microenvironment into consideration would have identified an additional 40 out of 290 patients (14%, premutation p = 0.001) with significantly worse outcomes (PFS, 95% CI 6-36, 49-73 versus 74-90 months) who were not identified by existing clinical (ISS stage III) and tumor (GEP-70) criteria as high risk. The main limitations of this study are that it relies on computationally identified cell types and that patients were treated with thalidomide rather than current therapies.

Conclusions: In this study, we observe that granulocyte signatures in the MM TME contribute to a more accurate prognosis. This implies that future researchers and clinicians treating patients should quantify TME components, in particular monocytes and granulocytes, which are often ignored in microenvironment studies.
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http://dx.doi.org/10.1371/journal.pmed.1003323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641353PMC
November 2020

No significant effect of caffeine on five kilometer running performance after muscle damage.

Int J Vitam Nutr Res 2020 Nov 4:1-9. Epub 2020 Nov 4.

Health and Physical Activity Building, Ball State University, Muncie, Indiana, USA.

Caffeine has documented hypoalgesic effects during exercise. However, there is a lack of research focusing on caffeine's potential analgesic effects to ameliorate delayed onset muscle soreness. A placebo controlled randomized cross-over trial was carried out to determine if 5 mg/kg of body weight (mg/kgBW) of caffeine attenuates muscle pain and improves 5 k running performance following delayed onset muscle soreness. Prior to participating, eleven runners (9 male; 2 female; age, 24.5 ± 6.3 years; height, 173.6 ± 7.8 cm; body mass, 66.3 ± 7.5 kg; BMI, 23.18 kg/m ± 1.6; VO 61.0 ± 6.1 ml/kg/min), were asked to discontinue supplement use for 72 hours and abstain from caffeine consumption for 48 hours. Participants performed a 30-minute downhill run on a treadmill set at -10% grade at 70% VO to induce delayed onset of muscle soreness. Participants then returned 48 hours after to complete a 5 k time trial run where they consumed either 5 mg/kgBW of caffeine or a placebo. Rate of perceived exertion and heart rate were taken every two minutes during the trial. There was no detectable statistical difference between 5 k performance between caffeine (1074.9 ± 119.7 sec) or placebo (1053.8 ± 86.8 sec) ( = .41). Algometer readings were similar between both treatments for muscle soreness in the rectus femoris ( = .791) and the vastus medialis oblique ( = .371). Muscle soreness ratings were found to be greater in the caffeine condition compared to the placebo condition ( = .030). There was no effect of treatment on rating of perceived exertion between conditions ( = .574). The present study suggests that caffeine is not effective at reducing muscle soreness, rating of perceived exertion, or improving running performance in a time trial in the presence of muscle soreness.
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http://dx.doi.org/10.1024/0300-9831/a000683DOI Listing
November 2020

A Fraction of Recommended Practices: Implementation of the FIFA 11+ in NCAA Soccer Programs.

Medicina (Kaunas) 2020 Aug 19;56(9). Epub 2020 Aug 19.

School of Kinesiology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.

National Collegiate Athletic Association (NCAA) soccer coaches implement numerous warm-up and flexibility strategies to prepare athletes for training and competition. The Fédération Internationale de Football Association (FIFA) developed the 11+ injury prevention program to reduce non-contact injuries. This study aimed to analyze the level of familiarity with and implementation of the evidence-based FIFA 11+ amongst NCAA Division I (DI) and Division III (DIII) men's and women's soccer coaches. NCAA soccer coaches in the United States received an Institutional Review Board-approved survey hyperlink. A total of 240 coaches completed the survey. The respondents represented 47.5% men's and 52.5% women's teams distributed within DI and DIII programs. Descriptive statistics are reported as frequency counts and mean ± standard deviation where applicable. Pearson's chi-square tests were performed to assess potential differences with a significance level set at α < 0.05. The results indicated that approximately 62% of the respondents reported being familiar with the FIFA 11+ program. Of those coaches familiar with the program, 15.0% reported full implementation, 57.5% reported partial implementation, and 27.5% reported no implementation. Chi-square analyses revealed significant differences in FIFA 11+ implementation based upon division level (χ = 4.56, = 0.033) and coaching certification levels (χ = 13.11, = 0.011). This study indicates that there is a gap between FIFA 11+ knowledge and actual implementation. To reduce the risk of non-contact injury, there is a need to educate coaches and athletic trainers on the purpose of the FIFA 11+ program and how to perform the exercises correctly.
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http://dx.doi.org/10.3390/medicina56090417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558407PMC
August 2020

Blood transcriptomic discrimination of bacterial and viral infections in the emergency department: a multi-cohort observational validation study.

BMC Med 2020 07 21;18(1):185. Epub 2020 Jul 21.

Division of Infection and Immunity, University College London, London, UK.

Background: There is an urgent need to develop biomarkers that stratify risk of bacterial infection in order to support antimicrobial stewardship in emergency hospital admissions.

Methods: We used computational machine learning to derive a rule-out blood transcriptomic signature of bacterial infection (SeptiCyte™ TRIAGE) from eight published case-control studies. We then validated this signature by itself in independent case-control data from more than 1500 samples in total, and in combination with our previously published signature for viral infections (SeptiCyte™ VIRUS) using pooled data from a further 1088 samples. Finally, we tested the performance of these signatures in a prospective observational cohort of emergency department (ED) patients with fever, and we used the combined SeptiCyte™ signature in a mixture modelling approach to estimate the prevalence of bacterial and viral infections in febrile ED patients without microbiological diagnoses.

Results: The combination of SeptiCyte™ TRIAGE with our published signature for viral infections (SeptiCyte™ VIRUS) discriminated bacterial and viral infections in febrile ED patients, with a receiver operating characteristic area under the curve of 0.95 (95% confidence interval 0.90-1), compared to 0.79 (0.68-0.91) for WCC and 0.73 (0.61-0.86) for CRP. At pre-test probabilities 0.35 and 0.72, the combined SeptiCyte™ score achieved a negative predictive value for bacterial infection of 0.97 (0.90-0.99) and 0.86 (0.64-0.96), compared to 0.90 (0.80-0.94) and 0.66 (0.48-0.79) for WCC and 0.88 (0.69-0.95) and 0.60 (0.31-0.72) for CRP. In a mixture modelling approach, the combined SeptiCyte™ score estimated that 24% of febrile ED cases receiving antibacterials without a microbiological diagnosis were due to viral infections. Our analysis also suggested that a proportion of patients with bacterial infection recovered without antibacterials.

Conclusions: Blood transcriptional biomarkers offer exciting opportunities to support precision antibacterial prescribing in ED and improve diagnostic classification of patients without microbiologically confirmed infections.
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http://dx.doi.org/10.1186/s12916-020-01653-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372897PMC
July 2020

Spectroscopic Investigation of Cysteamine Dioxygenase.

Biochemistry 2020 07 22;59(26):2450-2458. Epub 2020 Jun 22.

Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Thiol dioxygenases are mononuclear non-heme Fe-dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rather than the 2-His-1-carboxylate facial triad seen in most mononuclear non-heme Fe enzymes. A similar 3-His active site is found in the bacterial thiol dioxygenase 3-mercaptopropionate dioxygenase (MDO), which converts 3-mercaptopropionate into 3-sulfinopropionic acid as part of the bacterial sulfur metabolism pathway. In this study, we have investigated the active site geometric and electronic structures of a third non-heme Fe-dependent thiol dioxygenase, cysteamine dioxygenase (ADO), by using a spectroscopic approach. Although a 3-His facial triad had previously been implicated on the basis of sequence alignment and site-directed mutagenesis studies, little is currently known about the active site environment of ADO. Our magnetic circular dichroism and electron paramagnetic resonance data provide compelling evidence that ADO features a 3-His facial triad, like CDO and MDO. Despite this similar coordination environment, spectroscopic results obtained for ADO incubated with various substrate analogues are distinct from those obtained for the other Fe-dependent thiol dioxygenases. This finding suggests that the secondary coordination sphere of ADO is distinct from those of CDO and MDO, demonstrating the significant role that secondary-sphere residues play in dictating substrate specificity.
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http://dx.doi.org/10.1021/acs.biochem.0c00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717700PMC
July 2020

A bacterial biosynthetic pathway for methylated furan fatty acids.

J Biol Chem 2020 07 20;295(29):9786-9801. Epub 2020 May 20.

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA

Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in 2.4.1 and CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (1012)-11-methyloctadeca-10,12-dienoic acid (11Me-10,12-18:2), produced from (11)-methyloctadeca-11-enoic acid (11Me-12-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-1012-18:2 fatty acid phospholipid chain as a substrate. We discovered that also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.
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http://dx.doi.org/10.1074/jbc.RA120.013697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380195PMC
July 2020

Integrative network modeling reveals mechanisms underlying T cell exhaustion.

Sci Rep 2020 02 5;10(1):1915. Epub 2020 Feb 5.

Bristol-Myers Squibb, Summit, NJ, USA.

Failure to clear antigens causes CD8 T cells to become increasingly hypo-functional, a state known as exhaustion. We combined manually extracted information from published literature with gene expression data from diverse model systems to infer a set of molecular regulatory interactions that underpin exhaustion. Topological analysis and simulation modeling of the network suggests CD8 T cells undergo 2 major transitions in state following stimulation. The time cells spend in the earlier pro-memory/proliferative (PP) state is a fixed and inherent property of the network structure. Transition to the second state is necessary for exhaustion. Combining insights from network topology analysis and simulation modeling, we predict the extent to which each node in our network drives cells towards an exhausted state. We demonstrate the utility of our approach by experimentally testing the prediction that drug-induced interference with EZH2 function increases the proportion of pro-memory/proliferative cells in the early days post-activation.
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http://dx.doi.org/10.1038/s41598-020-58600-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002445PMC
February 2020

PreDSLpmo: A neural network-based prediction tool for functional annotation of lytic polysaccharide monooxygenases.

J Biotechnol 2020 Jan 9;308:148-155. Epub 2019 Dec 9.

Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh 173234, India. Electronic address:

Lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent oxidative enzymes, boost the degradation of polysaccharides such as cellulose, chitin, and others. While experimental methods are used to validate LPMO function, a computational method that can aid experimental methods and provide fast and accurate classification of sequences into LPMOs and its families would be an important step towards understanding the breadth of contributions these enzymes make in deconstruction of recalcitrant polysaccharides. In this study, we developed a machine learning-based tool called PreDSLpmo that employs two different approaches to functionally classify protein sequences into the major LPMO families (AA9 and AA10). The first approach uses a traditional neural network or multilayer percerptron-based approach, while the second employs bi-directional long short-term memory for sequence classification. Our method shows improvement in predictive power when compared with dbCAN2, an existing HMM-profile-based CAZyme predicting tool, on both validation and independent benchmark set.
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http://dx.doi.org/10.1016/j.jbiotec.2019.12.002DOI Listing
January 2020

Function-driven single-cell genomics uncovers cellulose-degrading bacteria from the rare biosphere.

ISME J 2020 03 21;14(3):659-675. Epub 2019 Nov 21.

U.S. Department of Energy, Joint Genome Institute, Walnut Creek, CA, 94598, USA.

Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus 'Cellulosimonas argentiregionis'. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.
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http://dx.doi.org/10.1038/s41396-019-0557-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031533PMC
March 2020

Extent and Origins of Functional Diversity in a Subfamily of Glycoside Hydrolases.

J Mol Biol 2019 03 25;431(6):1217-1233. Epub 2019 Jan 25.

Great Lakes Bioenergy Research Center, Madison, WI 53706 USA; Department of Biochemistry, University of Wisconsin, Madison, WI 53706 USA. Electronic address:

Some glycoside hydrolases have broad specificity for hydrolysis of glycosidic bonds, potentially increasing their functional utility and flexibility in physiological and industrial applications. To deepen the understanding of the structural and evolutionary driving forces underlying specificity patterns in glycoside hydrolase family 5, we quantitatively screened the activity of the catalytic core domains from subfamily 4 (GH5_4) and closely related enzymes on four substrates: lichenan, xylan, mannan, and xyloglucan. Phylogenetic analysis revealed that GH5_4 consists of three major clades, and one of these clades, referred to here as clade 3, displayed average specific activities of 4.2 and 1.2 U/mg on lichenan and xylan, approximately 1 order of magnitude larger than the average for active enzymes in clades 1 and 2. Enzymes in clade 3 also more consistently met assay detection thresholds for reaction with all four substrates. We also identified a subfamily-wide positive correlation between lichenase and xylanase activities, as well as a weaker relationship between lichenase and xyloglucanase. To connect these results to structural features, we used the structure of CelE from Hungateiclostridium thermocellum (PDB 4IM4) as an example clade 3 enzyme with activities on all four substrates. Comparison of the sequence and structure of this enzyme with others throughout GH5_4 and neighboring subfamilies reveals at least two residues (H149 and W203) that are linked to strong activity across the substrates. Placing GH5_4 in context with other related subfamilies, we highlight several possibilities for the ongoing evolutionary specialization of GH5_4 enzymes.
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http://dx.doi.org/10.1016/j.jmb.2019.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937791PMC
March 2019

Validation of a Host Response Assay, SeptiCyte LAB, for Discriminating Sepsis from Systemic Inflammatory Response Syndrome in the ICU.

Am J Respir Crit Care Med 2018 10;198(7):903-913

11 Immunexpress Inc., Seattle, Washington.

Rationale: A molecular test to distinguish between sepsis and systemic inflammation of noninfectious etiology could potentially have clinical utility.

Objectives: This study evaluated the diagnostic performance of a molecular host response assay (SeptiCyte LAB) designed to distinguish between sepsis and noninfectious systemic inflammation in critically ill adults.

Methods: The study employed a prospective, observational, noninterventional design and recruited a heterogeneous cohort of adult critical care patients from seven sites in the United States (n = 249). An additional group of 198 patients, recruited in the large MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium trial in the Netherlands ( www.clinicaltrials.gov identifier NCT01905033), was also tested and analyzed, making a grand total of 447 patients in our study. The performance of SeptiCyte LAB was compared with retrospective physician diagnosis by a panel of three experts.

Measurements And Main Results: In receiver operating characteristic curve analysis, SeptiCyte LAB had an estimated area under the curve of 0.82-0.89 for discriminating sepsis from noninfectious systemic inflammation. The relative likelihood of sepsis versus noninfectious systemic inflammation was found to increase with increasing test score (range, 0-10). In a forward logistic regression analysis, the diagnostic performance of the assay was improved only marginally when used in combination with other clinical and laboratory variables, including procalcitonin. The performance of the assay was not significantly affected by demographic variables, including age, sex, or race/ethnicity.

Conclusions: SeptiCyte LAB appears to be a promising diagnostic tool to complement physician assessment of infection likelihood in critically ill adult patients with systemic inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT01905033 and NCT02127502).
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http://dx.doi.org/10.1164/rccm.201712-2472OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835074PMC
October 2018

uses a Nu-class glutathione -transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin.

J Biol Chem 2018 04 15;293(14):4955-4968. Epub 2018 Feb 15.

From the Wisconsin Energy Institute,

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione -transferase NaGST is the only enzyme needed to remove glutathione from both ()- and ()-GS-HPV in We solved the crystal structure of NaGST and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr, Tyr, and Tyr) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from sp. SYK-6 (which can also break the β-aryl ether bond) and (which cannot break the β-aryl ether bond) can also cleave ()- and ()-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione -transferases.
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http://dx.doi.org/10.1074/jbc.RA117.001268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892560PMC
April 2018

Genome sequence of a diabetes-prone rodent reveals a mutation hotspot around the ParaHox gene cluster.

Proc Natl Acad Sci U S A 2017 07 3;114(29):7677-7682. Epub 2017 Jul 3.

Department of Zoology, University of Oxford, Oxford, OX1 3PS, United Kingdom;

The sand rat is a gerbil species native to deserts of North Africa and the Middle East, and is constrained in its ecology because high carbohydrate diets induce obesity and type II diabetes that, in extreme cases, can lead to pancreatic failure and death. We report the sequencing of the sand rat genome and discovery of an unusual, extensive, and mutationally biased GC-rich genomic domain. This highly divergent genomic region encompasses several functionally essential genes, and spans the ParaHox cluster which includes the insulin-regulating homeobox gene The sequence of sand rat has been grossly affected by GC-biased mutation, leading to the highest divergence observed for this gene across the Bilateria. In addition to genomic insights into restricted caloric intake in a desert species, the discovery of a localized chromosomal region subject to elevated mutation suggests that mutational heterogeneity within genomes could influence the course of evolution.
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http://dx.doi.org/10.1073/pnas.1702930114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530673PMC
July 2017

ACCP Template for Evaluating a Clinical Pharmacist.

Pharmacotherapy 2017 May 17;37(5):e21-e29. Epub 2017 Apr 17.

American College of Clinical Pharmacy, Lenexa, Kansas.

ACCP is committed to ensuring that clinical pharmacists possess the competencies necessary to deliver comprehensive medication management in team-based, direct patient care environments. These competencies are divided into six essential domains: direct patient care, pharmacotherapy knowledge, systems-based care and population health, communication, professionalism, and continuing professional development. The 2016 ACCP Clinical Practice Affairs Committee has developed an evaluation tool that includes the assessable tasks of today's clinical pharmacists that fall within each domain. This instrument can be used by institutions, organizations, and others responsible for clinical pharmacist performance evaluation and professional development.
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http://dx.doi.org/10.1002/phar.1927DOI Listing
May 2017

In-crystal reaction cycle of a toluene-bound diiron hydroxylase.

Nature 2017 04 27;544(7649):191-195. Epub 2017 Mar 27.

Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe/Fe species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O-O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.
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http://dx.doi.org/10.1038/nature21681DOI Listing
April 2017

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

J Biol Chem 2017 04 24;292(14):5932-5942. Epub 2017 Feb 24.

From the Department of Biochemistry and

There are more than 600 receptor-like kinases (RLKs) in , but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.
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http://dx.doi.org/10.1074/jbc.M116.761981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392584PMC
April 2017

Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling.

Biotechnol Biofuels 2017 2;10:31. Epub 2017 Feb 2.

US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.

Background: Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions.

Results: Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides.

Conclusions: The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.
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http://dx.doi.org/10.1186/s13068-017-0703-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288845PMC
February 2017

Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.

Proc Natl Acad Sci U S A 2016 12 30;113(50):14324-14329. Epub 2016 Nov 30.

Joint BioEnergy Institute, Emeryville, CA 94608;

Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 Å), its product vanillin (1.75 Å), and without any bound ligand (1.61 Å). The enzyme is a seven-bladed β-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4'-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.
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http://dx.doi.org/10.1073/pnas.1608917113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167157PMC
December 2016

Evolution and Ecology of Actinobacteria and Their Bioenergy Applications.

Annu Rev Microbiol 2016 09;70:235-54

Department of Bacteriology, University of Wisconsin-Madison, Wisconsin 53706; email:

The ancient phylum Actinobacteria is composed of phylogenetically and physiologically diverse bacteria that help Earth's ecosystems function. As free-living organisms and symbionts of herbivorous animals, Actinobacteria contribute to the global carbon cycle through the breakdown of plant biomass. In addition, they mediate community dynamics as producers of small molecules with diverse biological activities. Together, the evolution of high cellulolytic ability and diverse chemistry, shaped by their ecological roles in nature, make Actinobacteria a promising group for the bioenergy industry. Specifically, their enzymes can contribute to industrial-scale breakdown of cellulosic plant biomass into simple sugars that can then be converted into biofuels. Furthermore, harnessing their ability to biosynthesize a range of small molecules has potential for the production of specialty biofuels.
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http://dx.doi.org/10.1146/annurev-micro-102215-095748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703056PMC
September 2016

Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

PLoS Biol 2016 06 8;14(6):e1002475. Epub 2016 Jun 8.

DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.
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http://dx.doi.org/10.1371/journal.pbio.1002475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4898821PMC
June 2016

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

Protein Expr Purif 2016 10 6;126:33-41. Epub 2016 May 6.

Department of Biology, University of North Carolina at Chapel Hill, United States; Department of Pharmacology, University of North Carolina at Chapel Hill, United States. Electronic address:

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.
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http://dx.doi.org/10.1016/j.pep.2016.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594927PMC
October 2016

Depletion of regulatory T cells leads to an exacerbation of delayed-type hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade.

Dis Model Mech 2016 Apr 28;9(4):427-40. Epub 2016 Jan 28.

Department of Pharmacology, Zealand Pharma, Glostrup 2600, Denmark.

Rodent models of arthritis have been extensively used in the elucidation of rheumatoid arthritis (RA) pathogenesis and are instrumental in the development of therapeutic strategies. Here we utilise delayed-type hypersensitivity arthritis (DTHA), a model in C57BL/6 mice affecting one paw with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregsand the role of IL-17 in connection with Tregdepletion. Given the relevance of Tregsin RA, and the possibility of developing Treg-directed therapies, this approach could be relevant for advancing the understanding of Tregsin inflammatory arthritis. Selective depletion of Tregswas achieved using aFoxp3-DTR-eGFPmouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of theFoxp3gene. Anti-IL-17 monoclonal antibody (mAb) was used for IL-17 blockade. Numbers and activation of Tregsincreased in the paw and its draining lymph node in DTHA, and depletion of Tregsresulted in exacerbation of disease as shown by increased paw swelling, increased infiltration of inflammatory cells, increased bone remodelling and increased production of inflammatory mediators, as well as increased production of anti-citrullinated protein antibodies. Anti-IL-17 mAb treatment demonstrated that IL-17 is important for disease severity in both the presence and absence of Tregs, and that IL-17 blockade is able to rescue mice from the exacerbated disease caused by Tregdepletion and caused a reduction in RANKL, IL-6 and the number of neutrophils. We show that Tregsare important for the containment of inflammation and bone remodelling in DTHA. To our knowledge, this is the first study using theFoxp3-DTR-eGFPmouse on a C57BL/6 background for Tregdepletion in an arthritis model, and we here demonstrate the usefulness of the approach to study the role of Tregsand IL-17 in arthritis.
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http://dx.doi.org/10.1242/dmm.022905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852503PMC
April 2016

Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model.

Arthritis Res Ther 2016 Jan 23;18:28. Epub 2016 Jan 23.

Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin, Germany.

Background: The aims of the present study were to determine the relationship between bone destruction and bone formation in the delayed-type hypersensitivity arthritis (DTHA) model and to evaluate the effect of receptor activator of nuclear factor κB ligand (RANKL) blockade on severity of arthritis, bone destruction, and bone formation.

Methods: DTHA was induced in C57BL/6 mice. Inflammation, erosive joint damage, and new bone formation were semiquantitatively scored by histology. Osteoclast activity was assessed in vivo, and messenger RNA (mRNA) expression of mediators of bone destruction and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti-RANKL monoclonal antibody treatment was initiated at the time of immunization.

Results: Bone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction. Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling and a reduction of serum levels of SAP, MMP3, and CTX-I. Destruction of the subchondral bone was significantly reduced, while no effect on bone formation was seen.

Conclusions: Anti-RANKL treatment prevents joint destruction but does not prevent new bone formation in the DTHA model. Thus, although occurring sequentially during the course of DTHA, bone destruction and bone formation are apparently not linked in this model.
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http://dx.doi.org/10.1186/s13075-016-0931-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724155PMC
January 2016

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules.

Biotechnol Biofuels 2015 21;8:220. Epub 2015 Dec 21.

US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.

Background: Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.

Results: CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.

Conclusion: We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.
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http://dx.doi.org/10.1186/s13068-015-0402-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687162PMC
December 2015

Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.

Front Bioeng Biotechnol 2015 27;3:165. Epub 2015 Oct 27.

US Department of Energy Great Lakes Bioenergy Research Center , Madison, WI , USA ; Department of Biochemistry, University of Wisconsin-Madison , Madison, WI , USA.

Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.
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http://dx.doi.org/10.3389/fbioe.2015.00165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621489PMC
November 2015