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"Saturation mutagenesis"

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 catalyse 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 6, 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
Arti Tripathi, Kritika Gupta, Shruti Khare, Pankaj C Jain, Siddharth Patel, Prasanth Kumar, Ajai J Pulianmackal, Nilesh Aghera, Raghavan Varadarajan
Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility...
November 2016: Molecular Biology and Evolution
Xianliang Zheng, Bo Fang, Dongfei Han, Wenxia Yang, Feifei Qi, Hui Chen, Shengying Li
α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P...
2016: PloS One
Yutaro Baba, Jun-Ichi Sumitani, Kiyotaka Tanaka, Shuji Tani, Takashi Kawaguchi
Aspergillus aculeatus β-glucosidase 1 (AaBGL1) is one of the best cellobiose hydrolytic enzymes without transglycosylation products, among β-glucosidase from various origins, for use in cellulosic biomass conversion with Trichoderma cellulases. However, in our previous report, it was demonstrated that AaBGL1 has lower catalytic efficiency toward cellobiose, which is a major end product from cellulosic biomasses by Trichoderma reesei cellulases, than do gentiobiose and laminaribiose. Thus, we expected that there is room to enhance cellobiose hydrolytic activity of AaBGL1 by increasing catalytic efficiency (k cat/K m) up to that of gentiobiose or laminaribiose for accelerating the saccharification of cellulosic biomasses, and we performed site-saturation mutagenesis targeting nine amino acids supposed to constitute subsite +1 of AaBGL1...
July 21, 2016: Applied Microbiology and Biotechnology
Zhoutong Sun, Richard Lonsdale, Guangyue Li, Manfred T Reetz
Saturation mutagenesis at sites lining the binding pocket of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodology efforts, enabling comparisons between single code saturation mutagenesis (SCSM) and triple code saturation mutagenesis (TCSM) which employ only one versus three amino acids as building blocks, respectively...
July 13, 2016: Chembiochem: a European Journal of Chemical Biology
Michael A Stiffler, Subu K Subramanian, Victor H Salinas, Rama Ranganathan
Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example...
2016: Journal of Visualized Experiments: JoVE
James A Birrell, Christoph Laurich, Edward J Reijerse, Hideaki Ogata, Wolfgang Lubitz
Iron-sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron-sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions...
August 9, 2016: Biochemistry
Anna Małolepszy, Terry Mun, Niels Sandal, Vikas Gupta, Manu Dubin, Dorian Urbański, Niraj Shah, Asger Bachmann, Eigo Fukai, Hideki Hirakawa, Satoshi Tabata, Marcin Nadzieja, Katharina Markmann, Junyi Su, Yosuke Umehara, Takashi Soyano, Akira Miyahara, Shusei Sato, Makoto Hayashi, Jens Stougaard, Stig Uggerhøj Andersen
LTR retrotransposons are closely related to retroviruses and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640,653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) ( http : // lotus .au . dk). Insertion preferences are critical for effective gene targeting, and we exploit our large data set to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes...
June 20, 2016: Plant Journal: for Cell and Molecular Biology
Kang Zhou, Wei Zhao, Xiao-Qing Liu, Shu-Ming Li
The fungal indole prenyltransferase FtmPT1 is involved in the biosynthesis of fumitremorgins and catalyzes, in the presence of dimethylallyl diphosphate, a predominant regular prenylation of cyclo-L-Trp-L-Pro (brevianamide F) at position C-2 of the indole nucleus. Analysis of the substrate-bound structure of FtmPT1 revealed that brevianamide F forms a hydrogen bond via its carbonyl oxygen in the diketopiperazine moiety with the hydroxyl group of Tyr205 near the center of the prenyltransferase (PT) barrel. In this study, Tyr205 was mutated to 19 other proteinogenic amino acids by one-step site-directed mutagenesis...
June 16, 2016: Applied Microbiology and Biotechnology
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