Plant Nitrilases Research Articles

Biodegradation of nitriles derived from glucosinolates in rapeseed meal by BnNIT2: a nitrilase from Brassica napus with wide substrate specificity.

Nitriles derived from glucosinolates (GSLs) in rapeseed meal (RSM) can cause lesions on animal liver and kidneys. Nitrilase converts nitriles to carboxylic acids and NH3, eliminating their toxicity. Here we describe a nitrilase, BnNIT2, from Brassica napus (optimal temperature, 45°C; pH, 7.0) that is stable at 40°C and has a wide substrate specificity. Recombinant BnNIT2 converted the three main nitriles from GSLs (3-hydroxy-4-pentenenitrile, 3-butenenitrile, and 4-pentenenitrile), with the highest specific activity (58.6 U/mg) for 4-pentenenitrile. We used mutagenesis to improve the thermostability of BnNIT2; the resulting mutant BnNIT2-H90M had an ~ 14.5% increase in residual activity at 50°C for 1h. To verify the functionality of BnNIT2, GSLs were extracted from RSM and converted into nitriles at pH 5.0 in the presence of Fe2+. Then, BnNIT2 was used to degrade the nitriles from GSLs; ultimately, ~ 80% of nitriles were removed. Thus BnNIT2 is a potential enzyme for detoxification of RSM. KEY POINTS: • Functional identification of the plant nitrilase BnNIT2. • Identified a mutant, H90M, with improved thermostability. • BnNIT2 was capable of degrading nitriles from transformed GSLs.

Genetic and Functional Diversity of Nitrilases in Agaricomycotina.

Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant nitrilases and nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, β-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of nitrilase was determined for the first time.

Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure

Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.

Plant nitrilase: a new job for an old enzyme.

Nitrilases are versatile enzymes that hydrolyze nitriles to carboxylic acids and ammonia, but many members of this family lack defined biological functions. In plants, nitrilases have been associated with detoxification of cyanide-containing compounds and auxin biosynthesis; however, recent work suggests that the chemical versatility of these proteins contributes to metabolite repair. In this issue of the Biochemical Journal, Niehaus et al. demonstrate that the Nit1 nitrilase from Arabidopsis thaliana functions as a metabolite repair enzyme that removes deaminated glutathione from the cytoplasm and plastids.

Substrate specificity of plant nitrilase complexes is affected by their helical twist

Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant Capsella rubella, that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.

Arabidopsis NITRILASE 1 Contributes to the Regulation of Root Growth and Development through Modulation of Auxin Biosynthesis in Seedlings.

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). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of A. thaliana. We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, NIT2RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8

Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.

The function of the putative tumor suppressor Nit1 in lymphocytes (84.7)

Abstract The mammalian Nit1 gene is homologous to bacterial and plant nitrilases. In the fruit fly D. melanogaster and warms C. elegans, Nit1 is fused to the Fhit gene, whereas in mammals, FHIT and NIT1 are separate genes. FHIT is a tumor suppressor gene located at chromosome region 3p14.2, encompassing the common fragile site of the human genome. Somatic loss of Fhit in humans is associated with cancers in a wide variety of sites, including lung, kidney, stomach, pancreas, cervix, ovary, head and neck, breast, and hematopoetic cells. Proteins that engage in fusion events are expected to jointly participate in a biochemical or cellular pathway and/or to physically interact. Therefore, Nit1 may be functionally related to Fhit. Overexpression of Fhit and Nit1 can induce apoptosis. There was evidence showing that Fhit can induce FADD-mediated cell death. In this study, the in vivo function of Nit1 was analyzed using the Nit1 knockout mice. Various in vitro and in vivo analyses showed that Nit1 plays an important role in regulating cell cycle and proliferation in T lymphocytes.

Biological Functions of Mammalian Nit1, the Counterpart of the Invertebrate NitFhit Rosetta Stone Protein, a Possible Tumor Suppressor

The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.

Selective Hydrolysis of Aliphatic Dinitriles to Monocarboxylic Acids by a Nitrilase from Arabidopsis thaliana

The hydrolysis of a variety of dinitriles including α,ω-dicyanoalkanes 1, β-substituted glutaronitriles 5, and γ-cyanopimelonitrile 7 with a recombinant plant nitrilase from Arabidopsis thaliana, expressed in E. coli, is described. Conversion rate and selectivity of the hydrolysis of dinitriles 1a-f to ω-cyanocarboxylic acids 2a-f depend on the chain length. The enzyme activity markedly increases from malononitrile (1a) to octanedinitrile (1f). The selectivity, however, does not correlate with the rates. Up to a chain length of 6 C-atoms, the cyanocarboxylic acid is the only product, even at complete conversion of the starting material. Pimelonitrile (1e) is hydrolyzed to the cyanocarboxylic acid 2e without formation of diacid (<1%) up to 73% conversion. Glutaronitriles 5a-c were also hydrolyzed to the corresponding cyanobutanoic acids 6a-c with perfect selectivity. The nitrilase hydrolyzes exclusively the primary cyano group of 7 to give 3,5-dicyanoheptanoic acid (8a), whereby the selectivity is slightly reduced compared to the unsubstituted pimelonitrile (1e). If the hydrolysis is terminated at conversions ≤90%, pure 8a can be isolated in 72% yield (92% referred to conversion). After esterification of 8a to the methyl ester 8b, only the 5-cyano group but not the ester function was hydrolyzed enzymatically to give cyanoheptanedioic acid monoester (10).

Amino Acid Homologies between Human Biotinidase and Bacterial Aliphatic Amidases: Putative Identification of the Active Site of Biotinidase

A search of protein databases revealed amino acid homologies among human biotinidase, bacterial aliphatic amidases, and bacterial and plant nitrilases. Amino acids YRK210–212 of biotinidase are conserved among the enzyme families. This homology and naturally occurring mutations that cause biotinidase deficiency suggest that this region is essential for enzyme activity and is conserved from bacteria. Cys245 is likely the cysteine in the active site of biotinidase.

Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans.

The tumor suppressor gene FHIT encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study Fhit function we cloned and characterized FHIT genes from Drosophila melanogaster and Caenorhabditis elegans. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the D. melanogaster fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes: FHIT and NIT1, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine NIT1 genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine Fhit and Nit1 genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.