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
Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans-silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)-on M. vogae larvae at concentrations of 5 and 50 μM under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 μM, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 μM concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism.
- MeSH
- antioxidancia farmakologie MeSH
- hypoxie * MeSH
- larva účinky léků fyziologie MeSH
- Mesocestoides účinky léků fyziologie MeSH
- ochranné látky farmakologie MeSH
- silibinin farmakologie MeSH
- silymarin farmakologie MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
