Improved Fitness of <i>Arabidopsis thaliana</i> Nitrilase 2

Schreiner, Ulrike; Steinkellner, Georg; Rozzell, J. David; Glieder, Anton; Winkler, Margit

doi:10.1002/cctc.200900212

Cited by 10 publications

(10 citation statements)

References 26 publications

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“…In this study, we identified APX1 as a cultivar-related DAS with isoforms involved in different coexpression networks in the response to pathogen stress. NIT2 in A. thaliana is able to hydrolyze a broad substrate range [74], whereas a recent study revealed that AtNIT2 expression was de novo-induced by a plant pathogen (Pseudomonas syringae pv. tomato) and responsible for R gene-mediated resistance responses [75].…”

Section: Discussionmentioning

confidence: 98%

Differential Alternative Splicing Genes and Isoform Regulation Networks of Rapeseed (Brassica napus L.) Infected with Sclerotinia sclerotiorum

et al. 2020

Genes

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Alternative splicing (AS) is a post-transcriptional level of gene expression regulation that increases transcriptome and proteome diversity. How the AS landscape of rapeseed (Brassica napus L.) changes in response to the fungal pathogen Sclerotinia sclerotiorum is unknown. Here, we analyzed 18 RNA-seq libraries of mock-inoculated and S. sclerotiorum-inoculated susceptible and tolerant B. napus plants. We found that infection increased AS, with intron retention being the main AS event. To determine the key genes functioning in the AS response, we performed a differential AS (DAS) analysis. We identified 79 DAS genes, including those encoding splicing factors, defense response proteins, crucial transcription factors and enzymes. We generated coexpression networks based on the splicing isoforms, rather than the genes, to explore the genes’ diverse functions. Using this weighted gene coexpression network analysis alongside a gene ontology enrichment analysis, we identified 11 modules putatively involved in the pathogen defense response. Within these regulatory modules, six DAS genes (ascorbate peroxidase 1, ser/arg-rich protein 34a, unknown function 1138, nitrilase 2, v-atpase f, and amino acid transporter 1) were considered to encode key isoforms involved in the defense response. This study provides insight into the post-transcriptional response of B. napus to S. sclerotiorum infection.

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Section: Discussionmentioning

confidence: 98%

Differential Alternative Splicing Genes and Isoform Regulation Networks of Rapeseed (Brassica napus L.) Infected with Sclerotinia sclerotiorum

et al. 2020

Genes

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“…Although Steiner (1948) left open the possibility that Bz-CN was the active compound, he hypothesized that the hydrolysis of Bz-CN, probably inside the plant, generated the active PAA ( Steiner, 1948 ). As mentioned before, recombinant nitrilases of the NIT1-NIT3 subgroup accept various aliphatic and aromatic nitriles, including Bz-CN, as substrate in vitro ( Bartling et al, 1992 ; Vorwerk et al, 2001 ; Osswald et al, 2002 ; Ishikawa et al, 2007 ; Schreiner et al, 2010 ). It was previously hypothesized that PAA might be produced from benzylglucosinolate via myrosinase and nitrilase activity ( Ludwig-Müller and Cohen, 2002 ) but experimental data on in planta conversion of Bz-CN to PAA by nitrilases have to the best of our knowledge not yet been reported.…”

Section: Introductionmentioning

confidence: 93%

“…Arabidopsis possesses four nitrilase genes. NIT1 ( At3g44310 ), NIT2 ( At3g44300 ), and NIT3 ( At3g44320 ) encode enzymes with activity on GSL-derived nitriles when expressed in vitro ( Bartling et al, 1992 ; Vorwerk et al, 2001 ; Osswald et al, 2002 ; Schreiner et al, 2010 ). NIT4 ( At5g22300 ) encodes an enzyme with a strong substrate specificity for β-cyano- L -alanine, an intermediate in cyanide detoxification ( Piotrowski et al, 2001 ).…”

Section: Introductionmentioning

confidence: 99%

Benzyl Cyanide Leads to Auxin-Like Effects Through the Action of Nitrilases in Arabidopsis thaliana

2018

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Plants within the Brassicales order generate glucosinolate hydrolysis products that can exert different biological effects on several organisms. Here, we evaluated the physiological effects of one of these compounds, benzyl cyanide (phenylacetonitrile), when exogenously applied on Arabidopsis thaliana. Treatment with benzyl cyanide led to a dose-dependent reduction of primary root length and total biomass. Further morphological changes like elongated hypocotyls, epinastic cotyledons, and increased formation of adventitious roots resembled a severe auxin-overproducer phenotype. The elevated auxin response was confirmed by histochemical staining and gene expression analysis of auxin-responsive genes. Nitriles are converted by specific enzymes, nitrilases (NIT1-3), to their corresponding carboxylic acids. The nitrilase mutants nit1 and nit2 tolerated benzyl cyanide treatments better than the wild type, with nit2 being less resistant than nit1. A NIT2RNAi line suppressing several nitrilases was resistant to all tested benzyl cyanide concentrations. When exposed to phenylacetic acid (PAA) – the corresponding carboxylic acid of benzyl cyanide – wild type and mutant seedlings were, however, equally susceptible and showed a more severe auxin phenotype than upon cyanide treatment. Here, we demonstrate that the auxin-like effects triggered by benzyl cyanide on Arabidopsis are due to its nitrilase-mediated conversion to the natural auxin PAA.

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“…Although the nitrilase-catalyzed chemoenzymatic route for pregabalin from isobutylsuccinonitrile (IBSN) featured higher atom economy and lower cost, its application was restricted by either low activity or inferior enantioselectivity of the reported nitrilases (Xie et al, 2006;Zhang, Wu, Hao et al, 2019;Zhang, Wu, Liu et al, 2019). In addition, semi-rational and rational design of nitrilases were severely plagued by a deficiency of precise crystal structure information (Schreiner, Steinkellner, Rozzell, Glieder, & Winkler, 2010).…”

Section: Introductionmentioning

confidence: 99%

Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor

Zhang

et al. 2019

Biotech & Bioengineering

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Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi‐rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi‐rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4‐fold increase in whole‐cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes.

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Improved Fitness of Arabidopsis thaliana Nitrilase 2

Cited by 10 publications

References 26 publications

Differential Alternative Splicing Genes and Isoform Regulation Networks of Rapeseed (Brassica napus L.) Infected with Sclerotinia sclerotiorum

Differential Alternative Splicing Genes and Isoform Regulation Networks of Rapeseed (Brassica napus L.) Infected with Sclerotinia sclerotiorum

Benzyl Cyanide Leads to Auxin-Like Effects Through the Action of Nitrilases in Arabidopsis thaliana

Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor

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