Influence of mutation in the regulatory domain of α-isopropylmalate synthase from Saccharomyces cerevisiae on its activity and feedback inhibition.

Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in the l-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation of l-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding the LEU4 gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity by in vitro experiments. The results showed that not only the negative-feedback regulation by l-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence of l-leucine was significantly correlated with i-AmOH production.

Identification of Two Phenanthrene Derivatives from Australasian Allied Species in Genus Dendrobium.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.