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Alessio Peracchi, Maria Veiga-da-Cunha, Tomiko Kuhara, Kenneth W Ellens, Nicole Paczia, Vincent Stroobant, Agnieszka K Seliga, Simon Marlaire, Stephane Jaisson, Guido T Bommer, Jin Sun, Kay Huebner, Carole L Linster, Arthur J L Cooper, Emile Van Schaftingen
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i...
April 18, 2017: Proceedings of the National Academy of Sciences of the United States of America
Tawanda Zidenga, Dimuth Siritunga, Richard T Sayre
Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both β-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation...
2017: Frontiers in Plant Science
Jason M Park, B Trevor Sewell, Michael J Benedik
The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes...
April 2017: Applied Microbiology and Biotechnology
Felipe Acera, María Isabel Carmona, Francisco Castillo, Alberto Quesada, Rafael Blasco
Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a sole nitrogen source. Under this growth condition, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was encoded by nit4, one of the four nitrilase genes detected in the genome of this bacterium, and its expression in Escherichia coli enabled the recombinant strain to fully assimilate 3-cyanoalanine. P. pseudoalcaligenes CECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added...
May 1, 2017: Applied and Environmental Microbiology
Satya Prakash Chaurasia, Renu Deswal
The thiol-disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H2O2) induced changes in the gene expression have been reported, but efforts to detect H2O2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol-disulphide exchange in Brassica juncea seedlings following H2O2 (10 mM) treatment for 30 min...
February 2017: Protein Journal
Thomas Lehmann, Tim Janowitz, Beatriz Sánchez-Parra, Marta-Marina Pérez Alonso, Inga Trompetter, Markus Piotrowski, Stephan Pollmann
Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA)...
2017: Frontiers in Plant Science
Huayou Chen, Zhi Chen, Bangguo Wu, Jawad Ullah, Tianxi Zhang, Jinru Jia, Hongcheng Wang, Tianwei Tan
In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker...
2017: Journal of Molecular Microbiology and Biotechnology
Sam Mathew, Saravanan Prabhu Nadarajan, Uthayasuriya Sundaramoorthy, Hyunwoo Jeon, Taeowan Chung, Hyungdon Yun
OBJECTIVE: To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase. RESULTS: An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized...
April 2017: Biotechnology Letters
Lasse Janniche Nielsen, Peter Stuart, Martina Pičmanová, Simon Rasmussen, Carl Erik Olsen, Jesper Harholt, Birger Lindberg Møller, Nanna Bjarnholt
BACKGROUND: The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed. RESULTS: In this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses...
December 13, 2016: BMC Genomics
Ludmila Martínková, Lenka Rucká, Jan Nešvera, Miroslav Pátek
The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches...
January 2017: World Journal of Microbiology & Biotechnology
Zefan Li, Yuntao Zhu, Yuting Sun, Ke Qin, Weibing Liu, Wen Zhou, Xing Chen
Cell-selective protein metabolic labeling is of great interest for studying cell-cell communications and tissue homeostasis. We herein describe a nitrilase-activatable noncanonical amino acid tagging (NANCAT) strategy that exploits an exogenous nitrilase to enzymatically convert the nitrile-substituted precursors to their corresponding noncanonical amino acids (ncAAs), l-azidohomoalanine (Aha) or homopropargylglycine (Hpg), in living cells. Only cells expressing the nitrilase can generate Aha or Hpg in cellulo and metabolically incorporate them into the nascent proteins...
December 16, 2016: ACS Chemical Biology
Jizhe Sun, Huimin Yu, Jie Chen, Hui Luo, Zhongyao Shen
Rhodococcus ruber TH was selected as a parent strain to engineer for biomanufacturing of ammonium acrylate; the characteristics of this strain included accelerated growth rate, high cell tolerance and natively overexpressed nitrile hydratase (NHase). Transcriptome analysis revealed that the transcription levels of the native NHase, amidase and nitrilase were extremely high, moderate and extremely low, respectively. Through NHase-amidase double-knockout and amidase single-knockout, the engineered strains R. ruber THdAdN and R...
October 19, 2016: Journal of Industrial Microbiology & Biotechnology
Shuiqin Jiang, Lujia Zhang, Dongbin Cui, Zhiqiang Yao, Bei Gao, Jinping Lin, Dongzhi Wei
The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis...
October 6, 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 its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation...
December 28, 2016: Journal of Microbiology and Biotechnology
Mary A Crum, B Trevor Sewell, Michael J Benedik
Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied...
2016: Frontiers in Microbiology
Zhiqiang Yao, Lujia Zhang, Bei Gao, Dongbing Cui, Fengqing Wang, Xiao He, John Zenghui Zhang, Dong-Zhi Wei
The discovery of unique substrate is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming and expensive. Although in silico structure-based approaches show great promise, recent extensive studies show that these approaches remain a formidable challenge for current bio-computational methodologies. Here, we present an open-source, extensible and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of enzyme based on enzymes catalysis mechanism...
August 16, 2016: Journal of Chemical Information and Modeling
Chunyan Li, Zhenlei Yue, Fengzhao Feng, Chuanwu Xi, Hailian Zang, Xuejiao An, Keran Liu
There is a great need for efficient acetonitrile removal technology in wastewater treatment to reduce the discharge of this pollutant in untreated wastewater. In this study, a nitrilase gene (nit) isolated from a nitrile-degrading bacterium (Rhodococcus rhodochrous BX2) was cloned and transformed into a biofilm-forming bacterium (Bacillus subtilis N4) that expressed the recombinant protein upon isopropylthio-β-galactoside (IPTG) induction. The recombinant bacterium (B. subtilis N4-pHT01-nit) formed strong biofilms and had nitrile-degrading capability...
October 2016: Chemosphere
Lili Fu, Meng Wang, Bingying Han, Deguan Tan, Xuepiao Sun, Jiaming Zhang
Plant myrosinases (β-thioglucoside glucohydrolases) are classified into two subclasses, Myr I and Myr II. The biological function of Myr I has been characterized as a major biochemical defense against insect pests and pathogens in cruciferous plants. However, the biological function of Myr II remains obscure. We studied the function of two Myr II member genes AtTGG4 and AtTGG5 in Arabidopsis. RT-PCR showed that both genes were specifically expressed in roots. GUS-assay revealed that both genes were expressed in the root-tip but with difference: AtTGG4 was expressed in the elongation zone of the root-tip, while AtTGG5 was expressed in the whole root-tip...
June 7, 2016: International Journal of Molecular Sciences
Takuto Kumano, Yuko Takizawa, Sakayu Shimizu, Michihiko Kobayashi
One of the nitrile-synthesizing enzymes, β-cyano-L-alanine synthase, catalyzes β-cyano-L-alanine (β-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the β-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of β-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine...
September 12, 2016: Journal of General and Applied Microbiology
Du Seok Choi, Chae Woo Lim, Byung Kook Hwang
Proteomics and functional analyses of the Arabidopsis - Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection...
August 2016: Planta
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