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"Saturation mutagenesis"

Erik Eppinger, Andreas Stolz
The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C...
November 24, 2016: Protein Engineering, Design & Selection: PEDS
Yuki Honda, Qian Zang, Yasuhiro Shimizu, Mohammad Dadashipour, Zilian Zhang, Yutaka Kawarabayasi
: The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 Sugar-1-P NTase activity, nine double mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity...
November 18, 2016: Applied and Environmental Microbiology
Gülşah P Özgün, Emel B Ordu, H Esra Tütüncü, Emrah Yelboğa, Richard B Sessions, Nevin Gül Karagüler
In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD(+)-dependent cmFDH from NAD(+) to NADP(+) and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability...
2016: Scientifica
Xue-Cheng Jiao, Jiang Pan, Xu-Dong Kong, Jian-He Xu
A new high-throughput method for screening 2-deoxyribose-5-phosphate aldolase variants with a higher activity toward aldol reaction of unnatural aldehydes was established for the first time by coupling with an aldehyde dehydrogenase LeADH. The error-prone PCR and site-directed saturation mutagenesis libraries of aldolase LbDERA were constructed and screened using the high-throughput method. Two improved variants, LbDERAT29L and LbDERAF163Y, were identified and combined, giving a double mutant LbDERAT29L/F163Y which showed 7-fold higher activity than the native enzyme...
November 7, 2016: Biochemical and Biophysical Research Communications
Feng-Jiao Zhao, Yan Liu, Xiao-Qiong Pei, Chao Guo, Zhong-Liu Wu
(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity...
November 9, 2016: Applied Microbiology and Biotechnology
Wenqin Bai, Yufan Cao, Jun Liu, Qinhong Wang, Zhenhu Jia
BACKGROUND: Family 11 alkaline xylanases have great potential economic applications in the pulp and paper industry. In this study, we would improve the alkalophilicity of family 11 alkaline xylanase Xyn11A-LC from Bacillus sp. SN5, for the better application in this field. RESULTS: A random mutation library of Xyn11A-LC with about 10,000 clones was constructed by error-prone PCR. One mutant, M52-C10 (V116A and E135V), with improved alkalophilicity was obtained from the library...
November 8, 2016: BMC Biotechnology
Maria Duenas-Decamp, Li Jiang, Daniel Bolon, Paul R Clapham
The conformation of HIV-1 envelope (Env) glycoprotein trimers is key in ensuring protection against waves of neutralizing antibodies generated during infection, while maintaining sufficient exposure of the CD4 binding site (CD4bs) for viral entry. The CD4 binding loop on Env is an early contact site for CD4 while penetration of a proximal cavity by CD4 triggers Env conformational changes for entry. The role of residues in the CD4 binding loop in regulating the conformation of the trimer and trimer association domain (TAD) was investigated using a novel saturation mutagenesis approach...
November 2016: PLoS Pathogens
Yushen Du, Nicholas C Wu, Lin Jiang, Tianhao Zhang, Danyang Gong, Sara Shu, Ting-Ting Wu, Ren Sun
: Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods...
November 1, 2016: MBio
Amit R Majithia, Ben Tsuda, Maura Agostini, Keerthana Gnanapradeepan, Robert Rice, Gina Peloso, Kashyap A Patel, Xiaolan Zhang, Marjoleine F Broekema, Nick Patterson, Marc Duby, Ted Sharpe, Eric Kalkhoven, Evan D Rosen, Inês Barroso, Sian Ellard, Sekar Kathiresan, Stephen O'Rahilly, Krishna Chatterjee, Jose C Florez, Tarjei Mikkelsen, David B Savage, David Altshuler
Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions...
October 17, 2016: Nature Genetics
Monica Rodríguez-Bolaños, Nallely Cabrera, Ruy Perez-Montfort
The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T...
October 2016: Open Biology
Emily E Wrenbeck, Justin R Klesmith, James A Stapleton, Adebola Adeniran, Keith E J Tyo, Timothy A Whitehead
Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.
October 10, 2016: Nature Methods
Matthias Fink, Sarah Trunk, Mélanie Hall, Helmut Schwab, Kerstin Steiner
Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed...
2016: Frontiers in Microbiology
Naohiro Taniguchi, Hiroshi Murakami
Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning...
2017: Methods in Molecular Biology
Joseph J Maciag, Sarah H Mackenzie, Matthew B Tucker, Joshua L Schipper, Paul Swartz, A Clay Clark
The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2...
October 11, 2016: Proceedings of the National Academy of Sciences of the United States of America
Xiao-Fei Zhang, Guang-Yu Yang, Yong Zhang, Yuan Xie, Stephen G Withers, Yan Feng
The local flexibility of an enzyme's active center plays pivotal roles in catalysis, however, little is known about how the flexibility of these flexible residues affects stability. In this study, we proposed an active center stabilization (ACS) strategy to improve the kinetic thermostability of Candida rugosa lipase1. Based on the B-factor ranking at the region ~10 Å within the catalytic Ser209, 18 residues were selected for site-saturation mutagenesis. Based on three-tier high-throughput screening and ordered recombination mutagenesis, the mutant VarB3 (F344I/F434Y/F133Y/F121Y) was shown to be the most stable, with a 40-fold longer in half-life at 60 °C and a 12...
September 26, 2016: Scientific Reports
Chengtuo Niu, Linjiang Zhu, Xin Xu, Qi Li
Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures...
September 19, 2016: Applied Microbiology and Biotechnology
Irine Axarli, Abdi W Muleta, Evangelia G Chronopoulou, Anastassios C Papageorgiou, Nikolaos E Labrou
BACKGROUND: Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. METHODS: A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). RESULTS: Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes...
September 7, 2016: Biochimica et Biophysica Acta
Rui Huang, Hui Chen, Chao Zhong, Jae Eung Kim, Yi-Heng Percival Zhang
Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane...
2016: Scientific Reports
Jason M Park, Christian M Ponder, B Trevor Sewell, Michael J Benedik
Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large spiral shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from Bacillus pumilus, mutations in a predicted oligomeric surface region altered oligomerization and reduced activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants of that region which significantly perturbed oligomer formation...
September 2, 2016: Journal of Microbiology and Biotechnology
Molly Gasperini, Lea Starita, Jay Shendure
New technologies have recently enabled saturation mutagenesis and functional analysis of nearly all possible variants of regulatory elements or proteins of interest in single experiments. Here we discuss the past, present, and future of such multiplexed (functional) assays for variant effects (MAVEs). MAVEs provide detailed insight into sequence-function relationships, and they may prove critical for the prospective clinical interpretation of genetic variants.
October 2016: Nature Protocols
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