Gilvocarcin M

A combinatorial biosynthetic approach was used to interrogate the donor substrate flexibility of GilGT, the glycosyltransferase involved in C-glycosylation during gilvocarcin biosynthesis. Complementation of gilvocarcin mutant Streptomyces lividans TK24 (cosG9B3-U(-)), in which the biosynthesis of the natural sugar donor substrate was compromised, with various deoxysugar plasmids led to the generation of six gilvocarcin analogues with altered saccharide moieties. Characterization of the isolated gilvocarcin derivatives revealed five new compounds, including 4-β-C-D-olivosyl-gilvocarcin V (D-olivosyl GV), 4-β-C-D-olivosyl-gilvocarcin M (D-olivosyl GM), 4-β-C-D-olivosyl-gilvocarcin E (D-olivosyl GE), 4-α-C-L-rhamnosyl-gilvocarcin M (polycarcin M), 4-α-C-L-rhamnosyl-gilvocarcin E (polycarcin E), and the recently characterized 4-α-C-L-rhamnosyl-gilvocarcin V (polycarcin V). Preliminary anticancer assays showed that D-olivosyl-gilvocarcin and polycarcin V exhibit antitumor activities comparable to that of their parent drug congener, gilvocarcin V, against human lung cancer (H460), murine lung cancer (LL/2), and breast cancer (MCF-7) cell lines. Our findings demonstrate GilGT to be a moderately flexible C-glycosyltransferase able to transfer both D- and L-hexopyranose moieties to the unique angucyclinone-derived benzo[D]naphtho[1,2b]pyran-6-one backbone of the gilvocarcins.

The mode of action of gilvocarcins was studied. Gilvocarcins V, M and A possessed antibacterial activities decreasing in that order. Gilvocarcin V inhibited DNA synthesis in Bacillus subtilis through strong interaction with DNA and resulting cleavage. Gilvocarcin M showed interaction with DNA and a small change in DNA mobility upon electrophoresis in agarose gel, while gilvocarcin A showed no interaction with DNA, thus reflecting their relative biological activities.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.