Solubilization and Iterative Saturation Mutagenesis of α1,3-fucosyltransferase fromHelicobacter pylorito enhance its catalytic efficiency

2016 ◽  
Vol 113 (8) ◽  
pp. 1666-1675 ◽  
Author(s):  
Yun Hee Choi ◽  
Jong Hoon Kim ◽  
Bum Seok Park ◽  
Byung-Gee Kim
Keyword(s):  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Iterative Saturation Mutagenesis

scholarly journals Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

2015 ◽  
Vol 81 (8) ◽  
pp. 2919-2926 ◽  
Author(s):  
Chao Guo ◽  
Yanpu Chen ◽  
Yu Zheng ◽  
Wei Zhang ◽  
Yunwen Tao ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Significant Loss ◽  
Saturation Mutagenesis ◽  
Active Site Residues ◽  
Agrobacterium Radiobacter ◽  
Content Type ◽  
Halohydrin Dehalogenase ◽  
Aromatic Substrates ◽  
Chiral Epoxides ◽  
Iterative Saturation Mutagenesis

ABSTRACTHalohydrin dehalogenase fromAgrobacterium radiobacterAD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a highR-enantiopreference toward most aromatic substrates, whereas noS-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement inR-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from anERof 65 [WT] to anESof 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of itsR-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.

Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis

2009 ◽  
Vol 351 (18) ◽  
pp. 3287-3305 ◽  
Author(s):  
Despina J. Bougioukou ◽  
Sabrina Kille ◽  
Andreas Taglieber ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Enoate Reductase ◽  
Iterative Saturation Mutagenesis

scholarly journals Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 838 ◽  
Author(s):  
Raluca Bianca Tomoiagă ◽  
Souad Diana Tork ◽  
Ilka Horváth ◽  
Alina Filip ◽  
Levente Csaba Nagy ◽  
...  
Keyword(s):  
Rational Design ◽  
Stereoselective Synthesis ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
High Specificity ◽  
Saturation Mutagenesis ◽  
Petroselinum Crispum ◽  
Natural Substrate ◽  
Whole Cells ◽  
Pal Activity

Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).

Iterative Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods

2010 ◽  
Vol 132 (26) ◽  
pp. 9144-9152 ◽  
Author(s):  
Manfred T. Reetz ◽  
Shreenath Prasad ◽  
José D. Carballeira ◽  
Yosephine Gumulya ◽  
Marco Bocola
Keyword(s):  
Saturation Mutagenesis ◽  
Traditional Methods ◽  
Laboratory Evolution ◽  
Iterative Saturation Mutagenesis

scholarly journals Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

2012 ◽  
Vol 78 (18) ◽  
pp. 6647-6655 ◽  
Author(s):  
Yu Zhang ◽  
Jiao An ◽  
Wei Ye ◽  
Guangyu Yang ◽  
Zhi-Gang Qian ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Divergent Evolution ◽  
Laboratory Evolution ◽  
Content Type ◽  
Hydrolytic Activities ◽  
Geobacillus Kaustophilus ◽  
Amidohydrolase Superfamily

ABSTRACTThe phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates forin vitroevolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL fromGeobacillus kaustophilusHTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticideethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

Crystal structure and iterative saturation mutagenesis of ChKRED20 for expanded catalytic scope

2017 ◽  
Vol 101 (23-24) ◽  
pp. 8395-8404 ◽  
Author(s):  
Feng-Jiao Zhao ◽  
Yun Jin ◽  
Zhongchuan Liu ◽  
Chao Guo ◽  
Tong-Biao Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saturation Mutagenesis ◽  
Iterative Saturation Mutagenesis
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