The Rv3378c gene product from Mycobacterium tuberculosis encodes a
diterpene synthase to produce
tuberculosinol (3), 13R-isotuberculosinol (4a), and 13S-isotuberculosinol (4b) from tuberculosinyl
diphosphate (2). The product distribution ratios are 1 : 1 for 3 to 4 and 1 : 3 for 4a to 4b. The substrate specificity of the Rv3378c-encoded
enzyme was examined. The 3 labdadienyl
diphosphates,
copalyl diphosphate (
CDP) (7), ent-
CDP (8), and syn-
CDP (9), underwent the
conversion reaction, with good yields (67-78%).
Copalol (23) and
manool (24) were produced from 7, ent-
copalol (25) and ent-
manool (26) from 8, and syn-
copalol (27) and vitexifolin A (28) from 9. The ratio of 23 to 24 was 40 : 27, that of 25:26 was 22 : 50, and that of 27:28 was 16 : 62. Analysis on a GC-MS chromatograph equipped with a chiral column revealed that 24, 26, and 28 consisted of a mixture of 13R- (a) and 13S-stereoisomers (b) in the following ratio: ca. 1 : 1 for 24a to 24b, ca. 1 : 5 for 26a to 26b, and ca. 1 : 19 for 28a to 28b. The structures of these products indicate that the reactions of the 3 CDPs proceeded in the same fashion as that of 2. This is the first report on the enzymatic synthesis of natural
diterpenes manool, ent-
manool, and vitexifolin A. Both Rv3377c and Rv3378c genes are found in virulent Mycobacterium species, but not in avirulent species. We found that 3 and 4 inhibited the phagocytosis of opsonized
zymosan particles by human macrophage-like cells. Interestingly, the inhibitory activity was synergistically increased by the coexistence of 3 and 4b. Other
labdane-related
diterpenes, 13-16 and 23-28, had little or no inhibitory activity. This synergistic inhibition by 3 and 4 may provide further advantage to the impairment of phagocyte function, which might contribute to pathogenicity of M.
tuberculosis.