scholarly journals Improved the Activity of Phosphite Dehydrogenase and its Application in Plant Biotechnology

2021 ◽  
Vol 9 ◽  
Author(s):  
Tongtong Liu ◽  
Lili Yuan ◽  
Suren Deng ◽  
Xiangxian Zhang ◽  
Hongmei Cai ◽  
...  
Keyword(s):  
High Efficiency ◽  
Plant Biotechnology ◽  
Primary Source ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Saturation Mutagenesis ◽  
Nonrenewable Resource ◽  
Phosphite Dehydrogenase ◽  
Essential Prerequisite ◽  
P Fertilizers

Phosphorus (P) is a nonrenewable resource, which is one of the major challenges for sustainable agriculture. Although phosphite (Phi) can be absorbed by the plant cells through the Pi transporters, it cannot be metabolized by plant and unable to use as P fertilizers for crops. However, transgenic plants that overexpressed phosphite dehydrogenase (PtxD) from bacteria can utilize phosphite as the sole P source. In this study, we aimed to improve the catalytic efficiency of PtxD from Ralstonia sp.4506 (PtxDR4506), by directed evolution. Five mutations were generated by saturation mutagenesis at the 139th site of PtxD R4506 and showed higher catalytic efficiency than native PtxDR4506. The PtxDQ showed the highest catalytic efficiency (5.83-fold as compared to PtxDR4506) contributed by the 41.1% decrease in the Km and 2.5-fold increase in the kcat values. Overexpression of PtxDQ in Arabidopsis and rice showed increased efficiency of phosphite utilization and excellent development when phosphite was used as the primary source of P. High-efficiency PtxD transgenic plant is an essential prerequisite for future agricultural production using phosphite as P fertilizers.

scholarly journals High-efficiency retron-mediated single-stranded DNA production in plants

2021 ◽  
Author(s):  
Wenjun Jiang ◽  
Gundra Sivakrishna Rao ◽  
Rashid Aman ◽  
Haroon Butt ◽  
Radwa Kamel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Plant Biotechnology ◽  
Fold Increase ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Potential Applications

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

Enterocytes are the primary source of the chemokine ENA-78 in normal colon and ulcerative colitis

1997 ◽  
Vol 273 (1) ◽  
pp. G75-G82 ◽  
Author(s):  
S. Keates ◽  
A. C. Keates ◽  
E. Mizoguchi ◽  
A. Bhan ◽  
C. P. Kelly
Keyword(s):  
Ulcerative Colitis ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Primary Source ◽  
Fold Increase ◽  
Human Colon ◽  
Neutrophil Recruitment ◽  
Normal Colon ◽  
Necrosis Factor Alpha ◽  
Cytokine Stimulation

Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a neutrophil-directed C-X-C chemokine. We report that Caco-2 and T84 human intestinal epithelial cells produce ENA-78 after stimulation by interleukin (IL)-1 beta or tumor necrosis factor-alpha. Caco-2 cells show increased IL-8 production at 4-12 h and increased ENA-78 production at 8-24 h after cytokine stimulation. Immunohistochemical studies in normal human colon and in ulcerative colitis demonstrate ENA-78 immunoreactivity principally associated with crypt epithelial cells. Furthermore, human colonic tissues from patients with ulcerative colitis show elevated levels of ENA-78 mRNA (24-fold increase, P < 0.01) and protein (4-fold increase, P < 0.05) compared with normal controls. Thus ENA-78 is produced in normal colon and in ulcerative colitis and is predominantly of enterocyte origin. The kinetics of ENA-78 induction in human colon epithelial cell lines are delayed and prolonged compared with IL-8. We propose that ENA-78 and IL-8 serve complementary and sequential roles in neutrophil recruitment in ulcerative colitis. ENA-78 as an enterocyte-derived, neutrophil-activating chemokine may be especially important in neutrophil recruitment from the lamina propria into the epithelial layer.

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).

scholarly journals High Catalytic Efficiency of a Layered Coordination Polymer to Remove Simultaneous Sulfur and Nitrogen Compounds from Fuels

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 731
Author(s):  
Fátima Mirante ◽  
Ricardo F. Mendes ◽  
Filipe A. Almeida Paz ◽  
Salete S. Balula
Keyword(s):  
Coordination Polymer ◽  
High Efficiency ◽  
Catalytic Efficiency ◽  
Catalytic Performance ◽  
Nitrogen Compounds ◽  
One Pot ◽  
Extraction Solvent ◽  
Desulfurization Process ◽  
Morphological Differences ◽  
Model Diesel

An ionic lamellar coordination polymer based on a flexible triphosphonic acid linker, [Gd(H4nmp)(H2O)2]Cl2 H2O (1) (H6nmp stands for nitrilo(trimethylphosphonic) acid), presents high efficiency to remove sulfur and nitrogen pollutant compounds from model diesel. Its oxidative catalytic performance was investigated using single sulfur (1-BT, DBT, 4-MDBT and 4,6-DMDBT, 2350 ppm of S) and nitrogen (indole and quinolone, 400 ppm of N) model diesels and further, using multicomponent S/N model diesel. Different methodologies of preparation followed (microwave, one-pot, hydrothermal) originated small morphological differences that did not influenced the catalytic performance of catalyst. Complete desulfurization and denitrogenation were achieved after 2 h using single model diesels, an ionic liquid as extraction solvent ([BMIM]PF6) and H2O2 as oxidant. Simultaneous desulfurization and denitrogenation processes revealed that the nitrogen compounds are more easily removed from the diesel phase to the [BMIM]PF6 phase and consequently, faster oxidized than the sulfur compounds. The lamellar catalyst showed a high recycle capacity for desulfurization. The reusability of the diesel/H2O2/[BMIM]PF6 system catalyzed by lamellar catalyst was more efficient for denitrogenation than for desulfurization process using a multicomponent model diesel. This behavior is not associated with the catalyst performance but it is mainly due to the saturation of S/N compounds in the extraction phase.

Investigating the origin of high efficiency in confined multienzyme catalysis

Nanoscale ◽  
2019 ◽  
Vol 11 (45) ◽  
pp. 22108-22117 ◽  
Author(s):  
Yufei Cao ◽  
Xiaoyang Li ◽  
Jiarong Xiong ◽  
Licheng Wang ◽  
Li-Tang Yan ◽  
...  
Keyword(s):  
High Efficiency ◽  
Catalytic Efficiency ◽  
Enzyme Cascades

Biomimetic strategies have successfully been applied to confine multiple enzymes on scaffolds to obtain higher catalytic efficiency of enzyme cascades than freely distributed enzymes.

scholarly journals Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

2004 ◽  
Vol 279 (50) ◽  
pp. 52150-52159 ◽  
Author(s):  
Zhonghui Huang ◽  
Bo Zhou ◽  
Zhong-Yin Zhang
Keyword(s):  
Substrate Specificity ◽  
Protein Tyrosine Phosphatase ◽  
Cellular Proliferation ◽  
High Efficiency ◽  
Tyrosine Phosphatase ◽  
Substrate Recognition ◽  
Fold Increase ◽  
Phosphoryl Transfer ◽  
Protein Tyrosine ◽  
Hydrolysis Of

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183and Tyr185in the activation loop. Tyr185dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with akcat/Kmof 2.6 × 106m–1s–1. In contrast, thekcatK/mvalues for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis ofp-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (∼20-fold increase inkcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (∼20-fold increase inkcat/Km) by organizing the catalytic residues with respect to Tyr(P)185for optimal phosphoryl transfer.

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.

Identification of the metal center of chlorothalonil hydrolytic dehalogenase and enhancement of catalytic efficiency by directed evolution

2016 ◽  
Vol 1 (1) ◽  
pp. 30 ◽  
Author(s):  
Honghong Chen ◽  
Hui Wang ◽  
Tongtong Wang ◽  
Songren Huang ◽  
Xiaoxia Zang ◽  
...  
Keyword(s):  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Divalent Cations ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Atomic Emission ◽  
Luria Bertani ◽  
Metal Center ◽  
Emission Spectrometry ◽  
Plasma Atomic Emission Spectrometry

Chlorothalonil hydrolytic dehalogenase (Chd) is one of two reported hydrolytic dehalogenases for halogenated aromatics, and its catalysis is independent of coenzyme A and ATP. Earlier studies have established that the catalytic activity of Chd requires zinc ions. In this study, the metal center of Chd was systematically investigated. The metal content of Chd was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), and there were 2.14 equivalents of zinc/mol of protein, indicating that Chd contains a binuclear (Zn2+-Zn2+) center. It was found that other divalent cations, such as cobalt (Co2+) and cadmium (Cd2+), could substitute zinc (Zn2+) leading to relative activities of 91.6% and 120.0%, whereas manganese (Mn2+) and calcium (Ca2+) could substitute Zn2+ leading to relative activities of 29.1% and 57.0%, respectively. The enzymatic properties of these different metal ion-substituted Chd variants were also compared. Error-prone PCR and DNA shuffling methods were applied to directly evolve Chd to generate variants with higher catalytic efficiencies of chlorothalonil. Enhanced Chd variants were selected based on the formation of clear haloes on Luria-Bertani plates supplemented with chlorothalonil. One variant, Q146R/N168Y/S303G, exhibited a 4.43-fold increase in catalytic efficiency, showing the potential for application in the dehalogenation and detoxification of chlorothalonil contaminated-sites.

scholarly journals Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

2005 ◽  
Vol 387 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Clara E. CASES-GONZÁLEZ ◽  
Luis MENÉNDEZ-ARIAS
Keyword(s):  
Catalytic Efficiency ◽  
Fold Increase ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
Van Der Waals Interactions ◽  
Reverse Transcriptases ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Nucleotide Specificity ◽  
Hiv 1

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

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