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"Saturation mutagenesis"

Ge Qu, Richard Lonsdale, Peiyuan Yao, Guangyue Li, Beibei Liu, Manfred T Reetz, Zhoutong Sun
Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets...
January 4, 2018: Chembiochem: a European Journal of Chemical Biology
Almog Bregman-Cohen, Batel Deri, Shiran Maimon, Yael Pazy, Ayelet Fishman
2-Hydroxybiphenyl 3-monooxygenase (HbpA) is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing for the first time 3-hydroxybiphenyl, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity as the well-studied piceatannol...
January 3, 2018: Chembiochem: a European Journal of Chemical Biology
John S Chorba, Adri M Galvan, Kevan M Shokat
Proprotein convertase subtilisin/kexin type 9 (PCSK9) downregulates the low-density lipoprotein (LDL) receptor (LDL-R), elevating LDL cholesterol (LDL-C) and accelerating atherosclerotic heart disease, making it a promising cardiovascular drug target. To achieve its maximal effect on the LDL-R, PCSK9 requires auto-proteolysis. After cleavage, PCSK9 retains its prodomain in the active site as a self-inhibitor. Unlike other proprotein convertases, however, this retention is permanent, inhibiting any further protease activity for the remainder of its life cycle...
December 19, 2017: Journal of Biological Chemistry
Shengdong Ke, Vincent Anquetil, Jorge Rojas Zamalloa, Alisha Maity, Anthony Yang, Mauricio A Arias, Sergey Kalachikov, James J Russo, Jingyue Ju, Lawrence A Chasin
To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively...
December 14, 2017: Genome Research
Du-San Baek, Jeong-Ho Kim, Ye-Jin Kim, Yong-Sung Kim
Neuropilin-1 (NRP1), which functions as a co-receptor for vascular endothelial growth factor (VEGF) and is implicated in vascular permeability and tumorigenesis, has been targeted by peptides that specifically bind to the VEGF-binding region on NRP1. Like natural VEGF ligands, all known peptides with NRP1-binding activity bind only through a carboxy (C)-terminal R/K-x-x-R/K sequence motif (x stands for any amino acids); this strict requirement is called the C-end rule (CendR). Here, we report immunoglobulin Fc-fused NRP1-specific peptides deviating from CendR...
December 12, 2017: Molecular Pharmaceutics
Manfred T Reetz, Aitao Li, Carlos G Acevedo-Rocha, Zhoutong Sun, Tony Cox, Jia Lucy Xu
Saturation mutagenesis (SM) constitutes a widely used technique in directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Here we show how this fundamental problem can be solved by applying high-fidelity solid-phase chemical gene synthesis on Silicon-chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with stereoselective formation of (R,R)- and (S,S)-cyclohexane-1,2-diol...
November 24, 2017: Chembiochem: a European Journal of Chemical Biology
Shu-Ping Zou, Yu-Guo Zheng, Qun Wu, Zhi-Cai Wang, Ya-Ping Xue, Zhi-Qiang Liu
Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of (R)-epichlorohydrin...
November 18, 2017: Applied Microbiology and Biotechnology
Gur Pines, James D Winkler, Assaf Pines, Ryan T Gill
The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations...
November 14, 2017: MBio
Guangyue Li, Maximilian J L J Fürst, Hamid Reza Mansouri, Anna K Ressmann, Adriana Ilie, Florian Rudroff, Marko D Mihovilovic, Marco W Fraaije, Manfred T Reetz
Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S)...
November 13, 2017: Organic & Biomolecular Chemistry
Eunok Jung, Beom Gi Park, Hee-Wang Yoo, Joonwon Kim, Kwon-Young Choi, Byung-Gee Kim
CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively...
January 2018: Applied Microbiology and Biotechnology
Yanbing Zhu, Chaochao Qiao, Hebin Li, Lijun Li, Anfeng Xiao, Hui Ni, Zedong Jiang
This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7...
November 4, 2017: International Journal of Biological Macromolecules
Belén Infanzón, Pablo H Sotelo, Josefina Martínez, Pilar Diaz
Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/β-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X))...
January 2018: Enzyme and Microbial Technology
Thomas M Schmitt, David H Aggen, Kumiko Ishida-Tsubota, Sebastian Ochsenreither, David M Kranz, Philip D Greenberg
Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs...
November 6, 2017: Nature Biotechnology
Yuta Katano, Tongyang Li, Misato Baba, Miyo Nakamura, Masaaki Ito, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa
We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity...
November 6, 2017: Bioscience, Biotechnology, and Biochemistry
Arong Jung, Dhanarajan Rajakumar, Bong-June Yoon, Bradley J Baker
Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%...
October 2017: Experimental Neurobiology
Xueying Wang, Lei Wang, Xinping Lin, Xiaobing Yang, Wujun Liu, Zongbao K Zhao
Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus...
October 30, 2017: Applied Biochemistry and Biotechnology
Zhongyi Cheng, Lukasz Peplowski, Wenjing Cui, Yuanyuan Xia, Zhongmei Liu, Jialei Zhang, Michihiko Kobayashi, Zhemin Zhou
Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money- and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity towards rac-mandelonitrile (MAN)...
October 28, 2017: Biotechnology and Bioengineering
Leyuan Ma, Jeffrey I Boucher, Janet Paulsen, Sebastian Matuszewski, Christopher A Eide, Jianhong Ou, Garrett Eickelberg, Richard D Press, Lihua Julie Zhu, Brian J Druker, Susan Branford, Scot A Wolfe, Jeffrey D Jensen, Celia A Schiffer, Michael R Green, Daniel N Bolon
Developing tools to accurately predict the clinical prevalence of drug-resistant mutations is a key step toward generating more effective therapeutics. Here we describe a high-throughput CRISPR-Cas9-based saturated mutagenesis approach to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof of concept, we mutagenized a selected region within the leukemic oncogene BCR-ABL1 Using bulk competitions with a deep-sequencing readout, we analyzed hundreds of mutations under multiple drug conditions and found that the effects of mutations on growth in the presence or absence of drug were critical for predicting clinically relevant resistant mutations, many of which were cancer adaptive in the absence of drug pressure...
October 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
Feng-Jiao Zhao, Yun Jin, Zhongchuan Liu, Chao Guo, Tong-Biao Li, Zi-Yi Li, Ganggang Wang, Zhong-Liu Wu
ChKRED20 is an efficient and robust anti-Prelog ketoreductase that can catalyze the reduction of ketones to chiral alcohols as pharmaceutical intermediates with great industrial potential. To overcome its limitation on the bioreduction of ortho-substituted acetophenone derivatives, the X-ray crystal structure of the apo-enzyme of ChKRED20 was determined at a resolution of 1.85 Å and applied to the molecular modeling and reshaping of the catalytic cavity via three rounds of iterative saturation mutagenesis together with alanine scanning and recombination...
December 2017: Applied Microbiology and Biotechnology
Alexander Dennig, Alexandra Maria Weingartner, Tsvetan Kardashliev, Christina Andrea Müller, Erika Tassano, Martin Schürmann, Anna Joëlle Ruff, Ulrich Schwaneberg
The aromatic hydroxylation of pseudocumene (1a) and mesitylene (1b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1b (94%), whereas 1a was hydroxylated to a large extent on benzylic positions (46-64%). Site-saturation mutagenesis generated a new P450 BM3 mutant, herein named "variant M3" (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3 to 8-fold) and activity (75 to 230-fold) for 1a and 1b conversion...
October 9, 2017: Chemistry: a European Journal
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