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.
- MeSH
- aminohydrolasy chemie genetika metabolismus MeSH
- Basidiomycota klasifikace enzymologie genetika MeSH
- fumaráty metabolismus MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- fylogeneze MeSH
- kyanovodík metabolismus MeSH
- pyridiny metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study is to summarize the current progress in the design of biocatalytic processes applicable for the production of optically pure mandelic acids and their analogues. These compounds are used as building blocks for pharmaceutical chemistry and as chiral resolving agents. Their enzymatic syntheses mainly employed nitrile hydrolysis with nitrilases, ester hydrolysis, ammonolysis or esterification with lipases or esterases, and ketone reduction or alcohol oxidation with dehydrogenases. Each of these methods will be characterized in terms of its product concentrations, enantioselectivities, and the types of catalysts used. This review will focus on the dynamic kinetic resolution of mandelonitrile and analogues by nitrilases resulting in the production of high concentrations of (R)-mandelic acid or (R)-2-chloromandelic acid with excellent e.e. Currently, there is no comparable process for (S)-mandelic acids. However, the coupling of the S-selective cyanation of benzaldehyde with the enantioretentive hydrolysis of (S)-mandelonitrile thus obtained is a promising strategy. The major product can be changed from (S)-acid to (S)-amide using nitrilase mutants. The competitiveness of the biocatalytic and chemical processes will be assessed. This review covers the literature published within 2003-2017.
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. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.
- MeSH
- aminohydrolasy genetika metabolismus MeSH
- Bacteria enzymologie genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biodegradace MeSH
- biokatalýza MeSH
- databáze proteinů MeSH
- fungální proteiny genetika metabolismus MeSH
- houby enzymologie genetika MeSH
- metagenomika MeSH
- nitrily metabolismus MeSH
- proteinové inženýrství metody MeSH
- rekombinantní proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Potassium nitrate (E252) is widely used as a food preservative and has applications in the treatment of high blood pressure however high doses are carcinogenic. Larvae of Galleria mellonella were administered potassium nitrate to establish whether the acute effects in larvae correlated with those evident in mammals. Intra-haemocoel injection of potassium nitrate resulted in a significant increase in the density of circulating haemocytes and a small change in the relative proportions of haemocytes but haemocytes showed a reduced fungicidal ability. Potassium nitrate administration resulted in increased superoxide dismutase activity and in the abundance of a range of proteins associated with mitochondrial function (e.g. mitochondrial aldehyde dehydrogenase, putative mitochondrial Mn superoxide dismutase), metabolism (e.g. triosephosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase) and nitrate metabolism (e.g. aliphatic nitrilase, glutathione S-transferase). A strong correlation exists between the toxicity of a range of food preservatives when tested in G. mellonella larvae and rats. In this work a correlation between the effect of potassium nitrate in larvae and mammals is shown and opens the way to the utilization of insects for studying the in vivo acute and chronic toxicity of xenobiotics.
- MeSH
- aldehyddehydrogenasa metabolismus MeSH
- aminohydrolasy metabolismus MeSH
- dusičnany metabolismus farmakologie toxicita MeSH
- glutathiontransferasa metabolismus MeSH
- glyceraldehyd-3-fosfátdehydrogenasa (NADP+) metabolismus MeSH
- hemocyty účinky léků metabolismus MeSH
- hmyzí proteiny metabolismus MeSH
- katalasa metabolismus MeSH
- larva účinky léků metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- můry metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- proteom metabolismus MeSH
- proteomika MeSH
- sloučeniny draslíku farmakologie toxicita MeSH
- superoxiddismutasa metabolismus MeSH
- testy akutní toxicity metody MeSH
- triózafosfátizomeráza metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.
- MeSH
- aminohydrolasy genetika metabolismus MeSH
- data mining MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- klonování DNA MeSH
- nitrily metabolismus MeSH
- rekombinantní proteiny izolace a purifikace metabolismus MeSH
- substrátová specifita MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The recently discovered cytokinin (CK)-specific phosphoribohydrolase "Lonely Guy" (LOG) is a key enzyme of CK biosynthesis, converting inactive CK nucleotides into biologically active free bases. We have determined the crystal structures of LOG from Claviceps purpurea (cpLOG) and its complex with the enzymatic product phosphoribose. The structures reveal a dimeric arrangement of Rossmann folds, with the ligands bound to large pockets at the interface between cpLOG monomers. Structural comparisons highlight the homology of cpLOG to putative lysine decarboxylases. Extended sequence analysis enabled identification of a distinguishing LOG sequence signature. Taken together, our data suggest phosphoribohydrolase activity for several proteins of unknown function.
- MeSH
- aminohydrolasy chemie metabolismus MeSH
- Claviceps enzymologie MeSH
- cytokininy metabolismus MeSH
- fungální proteiny chemie metabolismus MeSH
- karboxylyasy chemie metabolismus MeSH
- molekulární modely * MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.
- MeSH
- aminohydrolasy chemie genetika metabolismus MeSH
- Arthrodermataceae enzymologie MeSH
- Aspergillus niger enzymologie MeSH
- druhová specificita MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- fylogeneze MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny mandlové metabolismus MeSH
- Nectria enzymologie MeSH
- Neurospora crassa enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.
- MeSH
- aldehydy metabolismus MeSH
- aminohydrolasy genetika metabolismus MeSH
- bakteriální proteiny chemie metabolismus MeSH
- cytokininy biosyntéza metabolismus MeSH
- interakce hostitele a patogenu MeSH
- mutace MeSH
- Mycobacterium tuberculosis účinky léků genetika metabolismus patogenita MeSH
- myši inbrední C57BL MeSH
- oxid dusnatý metabolismus farmakologie MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- suprese genetická MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Escherichia coli strains expressing different nitrilases transformed nitriles or KCN. Six nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetonitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising nitrilases for the transformation of industrially important substrates were found: the nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the nitrilases from N. crassa and A. niger for (R,S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine.
- MeSH
- aminohydrolasy chemie genetika metabolismus MeSH
- dehydratasy chemie genetika metabolismus MeSH
- Escherichia coli genetika MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- genom fungální * MeSH
- genomika MeSH
- houby enzymologie genetika MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
