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"Saturation mutagenesis"

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https://www.readbyqxmd.com/read/28530095/rational-design-of-bacillus-coagulans-nl01-l-arabinose-isomerase-and-using-its-f279i-variant-in-d-tagatose-production
#1
Zhaojuan Zheng, Wending Mei, Meijuan Xia, Qin He, Jia Ouyang
D-tagatose is a prospective functional sweetener that can be produced by L-arabinose isomerase (AI) from D-galactose. To improve the activity of AI toward D-galactose, the AI of Bacillus coagulans was rationally designed based on molecular modeling and docking. After alanine scanning and site-saturation mutagenesis, variant F279I that exhibited improved activity toward D-galactose was obtained. The optimal temperature and pH of F279I were determined to be 50°C and 8.0, respectively. This variant possessed 1...
May 22, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28526714/constans-imparts-dna-sequence-specificity-to-the-histone-fold-nf-yb-nf-yc-dimer
#2
Nerina Gnesutta, Roderick W Kumimoto, Swadhin Swain, Matteo Chiara, Chamindika Siriwardana, David Stephen Horner, Ben F Holt, Roberto Mantovani
NF-Y is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence-specificity. NF-YA shares a conserved domain with the CCT (CONSTANS, CONSTANS-LIKE, TOC1) proteins. We show that CONSTANS (CO/BBX1), a master flowering regulator, forms a trimer with Arabidopsis NF-YB2/NF-YC3 to efficiently bind the CORE element of the FT promoter. We term this complex NF-CO. Using saturation mutagenesis EMSAs and RNA-Seq profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site...
May 19, 2017: Plant Cell
https://www.readbyqxmd.com/read/28508385/improving-the-temperature-characteristics-and-catalytic-efficiency-of-a-mesophilic-xylanase-from-aspergillus-oryzae-aoxyn11a-by-iterative-mutagenesis-based-on-in-silico-design
#3
Xue-Qing Li, Qin Wu, Die Hu, Rui Wang, Yan Liu, Min-Chen Wu, Jian-Fang Li
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly(21) with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A(G21I), was selected from the mutagenesis library, E...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28504265/in-vitro-evolution-of-an-influenza-broadly-neutralizing-antibody-is-modulated-by-hemagglutinin-receptor-specificity
#4
Nicholas C Wu, Geramie Grande, Hannah L Turner, Andrew B Ward, Jia Xie, Richard A Lerner, Ian A Wilson
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs...
May 15, 2017: Nature Communications
https://www.readbyqxmd.com/read/28469274/crispr-cpf1-assisted-genome-editing-of-corynebacterium-glutamicum
#5
Yu Jiang, Fenghui Qian, Junjie Yang, Yingmiao Liu, Feng Dong, Chongmao Xu, Bingbing Sun, Biao Chen, Xiaoshu Xu, Yan Li, Renxiao Wang, Sheng Yang
Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%...
May 4, 2017: Nature Communications
https://www.readbyqxmd.com/read/28464395/specificity-effects-of-amino-acid-substitutions-in-promiscuous-hydrolases-context-dependence-of-catalytic-residue-contributions-to-local-fitness-landscapes-in-nearby-sequence-space
#6
Christopher D Bayer, Bert van Loo, Florian Hollfelder
Catalytic promiscuity can facilitate evolution of enzyme functions-a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site-saturating mutagenesis of an analogous, nucleophile-flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects...
May 2, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28464223/casting-eppcr-ceppcr-a-simple-random-mutagenesis-method-to-generate-high-quality-mutant-libraries
#7
Jianhua Yang, Anna J Ruff, Marcus Arlt, Ulrich Schwaneberg
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance...
May 2, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28425285/directed-evolution-of-carbonyl-reductase-from-rhodosporidium-toruloides-and-its-application-in-stereoselective-synthesis-of-tert-butyl-3r-5s-6-chloro-3-5-dihydroxyhexanoate
#8
Zhi-Qiang Liu, Lin Wu, Xiao-Jian Zhang, Ya-Ping Xue, Yu-Guo Zheng
tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3...
May 1, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28422960/predicting-the-impact-of-lynch-syndrome-causing-missense-mutations-from-structural-calculations
#9
Sofie V Nielsen, Amelie Stein, Alexander B Dinitzen, Elena Papaleo, Michael H Tatham, Esben G Poulsen, Maher M Kassem, Lene J Rasmussen, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen
Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells...
April 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28387245/design-and-application-of-a-lactulose-biosensor
#10
Jieyuan Wu, Peixia Jiang, Wei Chen, Dandan Xiong, Linglan Huang, Junying Jia, Yuanyuan Chen, Jian-Ming Jin, Shuang-Yan Tang
In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production...
April 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28381551/production-of-homogeneous-glycoprotein-with-multi-site-modifications-by-an-engineered-n-glycosyltransferase-mutant
#11
Qitao Song, Zhigang Wu, Yueyuan Fan, Woran Song, Peiru Zhang, Li Wang, Faxing Wang, Yangyang Xu, Peng George Wang, Jiansong Cheng
Naturally occurring N- glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study, we found that ApNGT, a newly discovered cytoplasmic N- glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity towards the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides...
April 5, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28284313/one-step-combined-focused-eppcr-and-saturation-mutagenesis-for-thermostability-evolution-of-a-new-cold-active-xylanase
#12
Juan Pablo Acevedo, Manfred T Reetz, Juan A Asenjo, Loreto P Parra
Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry...
May 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28255534/the-structure-of-toho1-%C3%AE-lactamase-in-complex-with-penicillin-reveals-the-role-of-tyr105-in-substrate-recognition
#13
Patricia S Langan, Venu Gopal Vandavasi, Kevin L Weiss, Jonathan B Cooper, Stephan L Ginell, Leighton Coates
The role of the conserved residue Tyr105 in class A β-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 β-lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring-opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site...
December 2016: FEBS Open Bio
https://www.readbyqxmd.com/read/28251763/a-13-week-research-based-biochemistry-laboratory-curriculum
#14
Scott T Lefurgy, Emily C Mundorff
Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given...
March 2, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28218758/variant-aware-saturating-mutagenesis-using-multiple-cas9-nucleases-identifies-regulatory-elements-at-trait-associated-loci
#15
Matthew C Canver, Samuel Lessard, Luca Pinello, Yuxuan Wu, Yann Ilboudo, Emily N Stern, Austen J Needleman, Frédéric Galactéros, Carlo Brugnara, Abdullah Kutlar, Colin McKenzie, Marvin Reid, Diane D Chen, Partha Pratim Das, Mitchel A Cole, Jing Zeng, Ryo Kurita, Yukio Nakamura, Guo-Cheng Yuan, Guillaume Lettre, Daniel E Bauer, Stuart H Orkin
Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants...
April 2017: Nature Genetics
https://www.readbyqxmd.com/read/28218303/a-combination-of-mutational-and-computational-scanning-guides-the-design-of-an-artificial-ligand-binding-controlled-lipase
#16
Marco Kaschner, Oliver Schillinger, Timo Fettweiss, Christina Nutschel, Frank Krause, Alexander Fulton, Birgit Strodel, Andreas Stadler, Karl-Erich Jaeger, Ulrich Krauss
Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae...
February 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28170244/structure-activity-relationship-and-molecular-mechanics-reveal-the-importance-of-ring-entropy-in-the-biosynthesis-and-activity-of-a-natural-product
#17
Hai L Tran, Katrina W Lexa, Olivier Julien, Travis S Young, Christopher T Walsh, Matthew P Jacobson, James A Wells
Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues...
February 22, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28165493/a-noise-trimming-and-positional-significance-of-transposon-insertion-system-to-identify-essential-genes-in-yersinia-pestis
#18
Zheng Rong Yang, Helen L Bullifent, Karen Moore, Konrad Paszkiewicz, Richard J Saint, Stephanie J Southern, Olivia L Champion, Nicola J Senior, Mitali Sarkar-Tyson, Petra C F Oyston, Timothy P Atkins, Richard W Titball
Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable...
February 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28163026/engineering-of-cyp106a2-for-steroid-9%C3%AE-and-6%C3%AE-hydroxylation
#19
Julia Nikolaus, Kim Thoa Nguyen, Cornelia Virus, Jan L Riehm, Michael Hutter, Rita Bernhardt
CYP 106A2 from Bacillus megaterium ATCC 13368 has been described as a 15β-hydroxylase showing also minor 11α-, 9α- and 6β-hydroxylase activity for progesterone conversion. Previously, mutant proteins with a changed selectivity towards 11α-OH-progesterone have already been produced. The challenge of this work was to create mutant proteins with a higher regioselectivity towards hydroxylation at positions 9 and 6 of the steroid molecule. 9α-hydroxyprogesterone exhibits pharmaceutical importance, because it is a useful intermediate in the production of physiologically active substances which possess progestational activity...
April 2017: Steroids
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
#20
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
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