RESUMO
A new microbial cyclic dipeptide (diketopiperazine), cyclo(D-Tyr-D-Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(D-Tyr-D-Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(D-Tyr-D-Phe) recorded significant antitumor activity against A549 cells (IC50 value: 10 µM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 µM. Cyclo(D-Tyr-D-Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(D-Tyr-D-Phe). Flow cytometry analysis showed that the cyclo(D-Tyr-D-Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase-3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(D-Tyr-D-Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(D-Tyr-D-Phe) with synthetic cyclo(D-Tyr-D-Phe) and cyclo(L-Tyr-L-Phe). Natural and synthetic cyclo(D-Tyr-D-Phe) recorded similar pattern of activity. Although synthetic cyclo(L-Tyr-L-Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(L-Tyr-L-Phe) recorded significant activity than natural and synthetic cyclo(D-Tyr-D-Phe). The results of the present study reveals that cyclo(D-Tyr-D-Phe) is more bioactive than cyclo(L-Tyr-L-Phe). To the best of our knowledge, this is the first time that cyclo(D-Tyr-D-Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(D-Tyr-D-Phe) is also reported for the first time.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Bacillus/química , Dipeptídeos/química , Dipeptídeos/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Dicetopiperazinas/química , Citometria de Fluxo , Humanos , Testes de Sensibilidade Microbiana , Microscopia de Contraste de Fase , Nematoides/microbiologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Proteus mirabilis/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Aminoacyl-tRNA-synthetases are crucial enzymes for initiation step of translation. Possessing editing activity, they protect living cells from misincorporation of non-cognate and non-proteinogenic amino acids into proteins. Tyrosyl-tRNA synthetase (TyrRS) does not have such editing properties, but it shares weak stereospecificity in recognition of d-/l-tyrosine (Tyr). Nevertheless, an additional enzyme, d-aminoacyl-tRNA-deacylase (DTD), exists to overcome these deficiencies. The precise catalytic role of hydroxyl groups of the tRNATyr A76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [32P]-labeled tRNATyr substrates have been tested in aminoacylation and deacylation assays. TyrRS demonstrates similar activity in charging the 2' and 3'-OH groups of A76 with l-Tyr. This synthetase can effectively use both OH groups as primary sites for aminoacylation with l-Tyr, but demonstrates severe preference toward 2'-OH, in charging with d-Tyr. In both cases, the catalysis is not substrate-assisted: neither the 2'-OH nor the 3'-OH group assists catalysis. In contrast, DTD catalyzes deacylation of d-Tyr-tRNATyr specifically from the 3'-OH group, while the 2'-OH assists in this hydrolysis.