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https://www.readbyqxmd.com/read/28520394/in-vitro-metabolic-engineering-of-amorpha-4-11-diene-biosynthesis-at-enhanced-rate-and-specific-yield-of-production
#1
Xixian Chen, Congqiang Zhang, Ruiyang Zou, Gregory Stephanopoulos, Heng-Phon Too
In vitro metabolic engineering is an alternative approach to cell-based biosynthesis. It offers unprecedented flexibility for the study of biochemical pathways, thus, providing useful information for the rational design and assembly of reaction modules. Herein, we took the advantage of in vitro metabolic engineering to initially gain insight into the regulatory network of a reconstituted amorpha-4,11-diene (AD) synthetic pathway. Guided by lin-log approximation, we rapidly identified the hitherto unrecognized inhibition of adenosine triphosphate (ATP) on both farnesyl pyrophosphate synthase (FPPS) and amorpha-4,11-diene synthase (ADS)...
May 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28429326/wax-ester-fermentation-and-its-application-for-biofuel-production
#2
Hiroshi Inui, Takahiro Ishikawa, Masahiro Tamoi
In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10-18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named "wax ester fermentation". The wax ester fermentation is quite unique in that the end products, i...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/28424264/identification-of-a-multienzyme-complex-for-glucose-metabolism-in-living-cells
#3
Casey L Kohnhorst, Minjoung Kyoung, Miji Jeon, Danielle L Schmitt, Erin L Kennedy, Julio Ramirez, Syrena M Bracey, Bao Tran Luu, Sarah J Russell, Songon An
Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags...
April 19, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28411640/expression-of-cellulolytic-enzyme-as-a-fusion-protein-that-reacts-specifically-with-a-polymeric-scaffold
#4
Priya Katyal, Yongkun Yang, Olga Vinogradova, Yao Lin
The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes built onto a common protein scaffold to facilitate the breakdown of the polymeric substrate, cellulose. One strategy for mimicking these highly effective nanomachines may involve the use of synthetic scaffolds that can react to and organize multiple engineered enzymes to promote synergistic action between the enzymes on the scaffold...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28393943/enhancing-coupled-enzymatic-activity-by-conjugating-one-enzyme-to-a-nanoparticle
#5
James N Vranish, Mario G Ancona, Eunkeu Oh, Kimihiro Susumu, Igor L Medintz
Enzymes have long been a prime research target for the commercial production of commodity and specialty chemicals, design of sensing devices, and the development of therapeutics and new chemical processes. Industrial applications for enzymes can potentially be enhanced by enzyme immobilization which often allows for increased enzyme stability, facile product purification, and minimized substrate diffusion times in multienzymatic cascades, but this is usually at the cost of a significant decrease in catalytic rates...
April 20, 2017: Nanoscale
https://www.readbyqxmd.com/read/28259128/directed-regulation-of-multienzyme-complexes-of-2-oxo-acid-dehydrogenases-using-phosphonate-and-phosphinate-analogs-of-2-oxo-acids
#6
REVIEW
A V Artiukhov, A V Graf, V I Bunik
2-Oxo acid dehydrogenase complexes are important metabolic checkpoints functioning at the intercept of sugar and amino acid degradation. This review presents a short summary of architectural, catalytic, and regulatory principles of the complexes structure and function, based on recent advances in studies of well-characterized family members. Special attention is given to use of synthetic phosphonate and phosphinate analogs of 2-oxo acids as selective and efficient inhibitors of the cognate complexes in biological systems of bacterial, plant, and animal origin...
December 2016: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/28203522/mass-spectral-determination-of-phosphopantetheinylation-specificity-for-carrier-proteins-in-mycobacterium-tuberculosis
#7
James Jung, Ghader Bashiri, Jodie M Johnston, Edward N Baker
Phosphopantetheinyl transferases (PPTases) are key elements in the modular syntheses performed by multienzyme systems such as polyketide synthases. PPTases transfer phosphopantetheine derivatives from Coenzyme A to carrier proteins (CPs), thus orchestrating substrate supply. We describe an efficient mass spectrometry-based protocol for determining CP specificity for a particular PPTase in organisms possessing several candidate PPTases. We show that the CPs MbtL and PpsC, both involved in synthesis of essential metabolites in Mycobacterium tuberculosis, are exclusively activated by the type 2 PPTase PptT and not the type 1 AcpS...
December 2016: FEBS Open Bio
https://www.readbyqxmd.com/read/28187530/structural-and-dynamic-characterization-of-a-freestanding-acyl-carrier-protein-involved-in-the-biosynthesis-of-cyclic-lipopeptide-antibiotics
#8
Subrata Paul, Hiroaki Ishida, Leonard T Nguyen, Zhihong Liu, Hans J Vogel
Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters...
May 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28181062/progress-in-biocatalysis-with-immobilized-viable-whole-cells-systems-development-reaction-engineering-and-applications
#9
REVIEW
Milan Polakovič, Juraj Švitel, Marek Bučko, Jaroslav Filip, Vilém Neděla, Marion B Ansorge-Schumacher, Peter Gemeiner
Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques...
February 8, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28159788/revisiting-the-regulation-of-the-primary-scaffoldin-gene-in-clostridium-thermocellum
#10
Lizett Ortiz de Ora, Iván Muñoz-Gutiérrez, Edward A Bayer, Yuval Shoham, Raphael Lamed, Ilya Borovok
Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σ(I) factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum In the present report, we provide experimental evidence demonstrating that the C...
April 15, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28138383/systematic-chemoenzymatic-synthesis-of-o-sulfated-sialyl-lewis-x-antigens
#11
Abhishek Santra, Hai Yu, Nova Tasnima, Musleh M Muthana, Yanhong Li, Jie Zeng, Nicholas J Kenyond, Angelique Y Louie, Xi Chen
O-Sulfated sialyl Lewis x antigens play important roles in nature. However, due to their structural complexity, they are not readily accessible by either chemical or enzymatic synthetic processes. Taking advantage of a bacterial sialyltransferase mutant that can catalyze the transfer of different sialic acid forms from the corresponding sugar nucleotide donors to Lewis x antigens which are fucosylated glycans as well as an efficient one-pot multienzyme (OPME) sialylation system, O-sulfated sialyl Lewis x antigens containing different sialic acid forms and O-sulfation at different locations were systematically synthesized by chemoenzymatic methods...
April 1, 2016: Chemical Science
https://www.readbyqxmd.com/read/28134951/converting-pasteurella-multocida%C3%AE-2-3-sialyltransferase-1-pmst1-to-a-regioselective-%C3%AE-2-6-sialyltransferase-by-saturation-mutagenesis-and-regioselective-screening
#12
John B McArthur, Hai Yu, Jie Zeng, Xi Chen
A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1...
February 21, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28120367/photoelectrochemical-reduction-of-carbon-dioxide-to-methanol-through-a-highly-efficient-enzyme-cascade
#13
Su Keun Kuk, Raushan K Singh, Dong Heon Nam, Ranjitha Singh, Jung-Kul Lee, Chan Beum Park
Natural photosynthesis is an effective route for the clean and sustainable conversion of CO2 into high-energy chemicals. Inspired by the natural process, a tandem photoelectrochemical (PEC) cell with an integrated enzyme-cascade (TPIEC) system was designed, which transfers photogenerated electrons to a multienzyme cascade for the biocatalyzed reduction of CO2 to methanol. A hematite photoanode and a bismuth ferrite photocathode were applied to fabricate the iron oxide based tandem PEC cell for visible-light-assisted regeneration of the nicotinamide cofactor (NADH)...
January 25, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28107425/using-an-inducible-promoter-of-a-gene-encoding-penicillium-verruculosum-glucoamylase-for-production-of-enzyme-preparations-with-enhanced-cellulase-performance
#14
Alexander G Bulakhov, Pavel V Volkov, Aleksandra M Rozhkova, Alexander V Gusakov, Vitaly A Nemashkalov, Aidar D Satrutdinov, Arkady P Sinitsyn
BACKGROUND: Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. RESULTS: Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P...
2017: PloS One
https://www.readbyqxmd.com/read/27925306/graphene-oxide-based-enzyme-nanoarchitectonics-for-substrate-channeling
#15
Motilal Mathesh, Jingquan Liu, Colin J Barrow, Wenrong Yang
The controlled spatial organization or compartmentalization of multi-enzyme cascade reactions to transfer a substrate from one enzyme to another for substrate channeling on scaffolds has sparked increasing interest in recent years. Here, we use graphene oxides to study the dependence of the activity of cascade reactions in a closely packed, randomly immobilized enzyme system on a 2 D scaffold. We first observe that the hydrophobicity of graphene oxides and various enzyme architectures for co-immobilized systems are important attributes for achieving high product-conversion rates...
December 7, 2016: Chemistry: a European Journal
https://www.readbyqxmd.com/read/27806579/cerium-oxide-based-nanozymes-redox-phenomenon-at-biointerfaces
#16
Sanjay Singh
Cerium oxide nanoparticles (nanoceria) are known to exhibit enzymelike activity, such as biological catalase, oxidase, superoxide dismutase, and peroxidase enzymelike activities. Catalytically active nanoceria offer several advantages over natural enzymes, such as controlled synthesis at low cost, tunable catalytic activities, as well as high stability against stringent physiological conditions. Exploiting these properties, several biomedical applications, such as biosensing, immunoassay, drug delivery, radiation protection, and tissue engineering, have been maneuvered...
November 2, 2016: Biointerphases
https://www.readbyqxmd.com/read/27759119/enzymatic-diversity-of-the-clostridium-thermocellum-cellulosome-is-crucial-for-the-degradation-of-crystalline-cellulose-and-plant-biomass
#17
Katsuaki Hirano, Masahiro Kurosaki, Satoshi Nihei, Hiroki Hasegawa, Suguru Shinoda, Mitsuru Haruki, Nobutaka Hirano
The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified...
October 19, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27736072/sequential-one-pot-multienzyme-chemoenzymatic-synthesis-of-glycosphingolipid-glycans
#18
Hai Yu, Yanhong Li, Jie Zeng, Vireak Thon, Dung M Nguyen, Thao Ly, Hui Yu Kuang, Alice Ngo, Xi Chen
Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe)...
November 18, 2016: Journal of Organic Chemistry
https://www.readbyqxmd.com/read/27720939/differential-expression-of-micrornas-from-mir-17-family-in-the-cerebellum-of-mucopolysaccharidosis-type-i-mice
#19
Vanessa Gonçalves Pereira, Matheus Trovão Queiroz, Vânia D'Almeida
Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase, involved in degradation of glycosaminoglycans. Clinical manifestations are widely variable and patients with severe phenotype present developmental delay and cognitive decline, among other systemic alterations. MPS I patients present secondary accumulation of gangliosides in neuronal cells, besides accumulation of undegraded glycosaminoglycans. Reduction of Neu1 expression has been previously observed in the cerebellum of MPS I mice; to be active, neuraminidase 1 forms the lysosomal multienzyme complex (LMC) with two other proteins, β-galactosidase and protective protein/cathepsin A, involved in stepwise degradation of gangliosides in the lysosomes...
December 31, 2016: Gene
https://www.readbyqxmd.com/read/27624811/a-rapid-and-efficient-luminescence-based-method-for-assaying-phosphoglycosyltransferase-enzymes
#20
Debasis Das, Marthe T C Walvoort, Vinita Lukose, Barbara Imperiali
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays...
September 14, 2016: Scientific Reports
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