Almost 100 genes within the genus Bradyrhizobium are known to potentially encode aldoxime dehydratases (Oxds), but none of the corresponding proteins have been characterized yet. Aldoximes are natural substances involved in plant defense and auxin synthesis, and Oxds are components of enzymatic cascades enabling bacteria to transform, utilize and detoxify them. The aim of this work was to characterize a representative of the highly conserved Oxds in Bradyrhizobium spp. which include both plant symbionts and members of the soil communities. The selected oxd gene from Bradyrhizobium sp. LTSPM299 was expressed in Escherichia coli, and the corresponding gene product (OxdBr1; GenBank: WP_044589203) was obtained as an N-His6-tagged protein (monomer, 40.7 kDa) with 30-47% identity to Oxds characterized previously. OxdBr1 was most stable at pH ca. 7.0-8.0 and at up to 30 °C. As substrates, the enzyme acted on (aryl)aliphatic aldoximes such as E/Z-phenylacetaldoxime, E/Z-2-phenylpropionaldoxime, E/Z-3-phenylpropionaldoxime, E/Z-indole-3-acetaldoxime, E/Z-propionaldoxime, E/Z-butyraldoxime, E/Z-valeraldoxime and E/Z-isovaleraldoxime. Some of the reaction products of OxdBr1 are substrates of nitrilases occurring in the same genus. Regions upstream of the oxd gene contained genes encoding a putative aliphatic nitrilase and its transcriptional activator, indicating the participation of OxdBr1 in the metabolic route from aldoximes to carboxylic acids.
The purpose of this study is to summarize the current knowledge of the enzymes which are involved in the hydrolysis of cyanide, i.e., cyanide hydratases (CHTs; EC 4.2.1.66) and cyanide dihydratases (CynD; EC 3.5.5.1). CHTs are probably exclusively produced by filamentous fungi and widely occur in these organisms; in contrast, CynDs were only found in a few bacterial genera. CHTs differ from CynDs in their reaction products (formamide vs. formic acid and ammonia, respectively). Several CHTs were also found to transform nitriles but with lower relative activities compared to HCN. Mutants of CynDs and CHTs were constructed to study the structure-activity relationships in these enzymes or to improve their catalytic properties. The effect of the C-terminal part of the protein on the enzyme activity was determined by constructing the corresponding deletion mutants. CynDs are less active at alkaline pH than CHTs. To improve its bioremediation potential, CynD from Bacillus pumilus was engineered by directed evolution combined with site-directed mutagenesis, and its operation at pH 10 was thus enabled. Some of the enzymes have been tested for their potential to eliminate cyanide from cyanide-containing wastewaters. CynDs were also used to construct cyanide biosensors.
- MeSH
- Bacteria enzymologie MeSH
- biosenzitivní techniky * MeSH
- biotransformace MeSH
- dehydratasy chemie genetika metabolismus MeSH
- houby enzymologie MeSH
- hydrolasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- koncentrace vodíkových iontů MeSH
- kyanidy analýza metabolismus MeSH
- látky znečišťující životní prostředí analýza metabolismus MeSH
- mutační analýza DNA MeSH
- mutantní proteiny genetika metabolismus MeSH
- proteinové inženýrství MeSH
- stabilita enzymů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy 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
Nutritional quality of human and animal foodstuffs is determined by the content of essential amino acids. Barley is the fourth most important cereal of the world and the second most important cereal grown in the Czech Republic. Cereal grains such as barley contain insufficient levels of some essential amino acids, especially lysine. Dihydrodipicolinate synthase is the key enzyme involved in the regulatory step for lysine biosynthesis. Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA gene from Escherichia coli coding for the bacterial dihydrodipicolinate synthase were used for transformation of barley. An Agrobacterium-mediated technique was used for transformation of immature embryos of spring barley cv. Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by PCR, real-time PCR, gel electrophoresis, and Western blot. Amino acid content was analyzed by HPLC after HCl hydrolysis. The lysine content in leaves of the T1 generation plant no. 5/5 was 50% higher than in wild-type plants; the lysine content in seeds of T2 generation plant no. 5/16 was 30% higher than in wild-type seeds of spring barley cv. Golden Promise.
- MeSH
- aminokyseliny analýza metabolismus MeSH
- chromatografie iontoměničová MeSH
- dehydratasy biosyntéza genetika metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- geneticky modifikované rostliny chemie enzymologie genetika MeSH
- hydrolýza MeSH
- ječmen (rod) chemie enzymologie genetika MeSH
- listy rostlin chemie MeSH
- lysin analýza metabolismus MeSH
- polymerázová řetězová reakce MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- dehydratasy genetika nedostatek MeSH
- dítě MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- meglutol moč MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- kazuistiky MeSH
