The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile-->Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter only occurs when the protein is at the recognition site. This contrasts to wild-type EcoRV, where Mn2+ ions bind readily to complexes with either cognate and noncognate DNA and only Mg2+ shows the discrimination between the complexes. The structural perturbation is a specific consequence of leucine in place of isoleucine, since mutants with valine or alanine were similar to wild-type EcoRV.
Natural products with non-toxic and environmentally friendly properties are good resources for skin-whitening cosmetic agents when compared to artificial synthetic chemicals. Here, we investigated the effect of glyceollin produced to induce disease resistance responses of soybean to specific races of an incompatible pathogen, phytophthora sojae, on melanogenesis and discussed their mechanisms in melanin biosynthesis. We found that glyceollin inhibits melanin synthesis and tyrosinase activity in B16 melanoma cells without cytotoxicity. To elucidate the mechanism of the effect of glyceollin on melanogenesis, we conducted western blot analysis for melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2. Glyceollin inhibited tyrosinase and TRP-1 protein expression. Additionally, glyceollin effectively inhibited intracellular cAMP levels in B16 melanoma cells stimulated by α-melanocyte stimulating hormone (α-MSH). These results suggest that the whitening activity of glyceollin may be due to the inhibition of cAMP involved in the signal pathway of α-MSH in B16 melanoma cells. [BMB reports 2010; 43(7): 461-467]
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