Figure 3 HPLC analysis of biochemical assays of LobP1 and the scheme for LobP1-catalyzed hydroxylation. A typical assay was performed in PBS buffer (50 mM, pH 7.6) at 30 ℃ for 1 h, containing the substrate 400 μ M, LobP1 1.8 μ M, Fdx/FdR 5 μ M each, NADPH 2 mM. Representative nonsubstrates (boxed) of LobP1 were also shown. Notably, although LobP1 could not recognize 11 , 13 , and 14 as substrates for C-32 hydroxylation, in vivoproduction of their hydroxylated counterparts 18 , 16 , and 17 , respectively, was observed (see Figure 2).
Antibacterial and cytotoxic activity evaluation of LOBs
LOBs obtained in this study were also evaluated for antibacterial activities against Straphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus (MRSA) shhs-A1,Bacillus subtilis 1064 and Micrococcus luteus SCSIO ML01, and the results were summarized in Table 2. All the tested LOBs displayed significant activities against B. subtilis 1064 andM. luteus SCSIO ML01 with MIC values ranging from 0.125 to 8μ g mL-1. Compounds 18 showed no activity or weak activities against S. aureus ATCC 29213 and MRSA, while compound 6 exhibited antibacterial activities against these two strains with MIC values of 16 μ g mL-1. And compound 7 showed better antibacterial activities than its corresponding hydroxylated product1 . In addition, Compounds 13 and57 were tested for the cytotoxic activities against four human tumor cell lines: SF-268, MCF-7, HeG-2 and A549 (Table 2). Notably, compound 6 displayed overall better cytotoxic activities than other tested compounds, including its hydroxylated product 2 , with IC50 values of 11.94, 12.07, 3.11 and 4.34 μ M against SF-268, MCF-7, HeG-2 and A549, respectively.
Table 2 Antibacterial and cytotoxic activities of compounds1 -8