title: Substrate specificity of Rv3378c, an enzyme from Mycobacterium tuberculosis, and the inhibitory activity of the bicyclic diterpenoids against macrophage phagocytosis.
abstract: 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.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture and Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan. hoshitsu@agr.niigata-u.ac.jp
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Hoshino
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nakano
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ootsuka
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Takashi
authors.initials: T
authors.lastname: Hara
mesh_terms: D055162:Biocatalysis; D001643:Bridged Bicyclo Compounds / Q000737:chemistry* / Q000494:pharmacology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004224:Diterpenes / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000187:drug effects*; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010587:Phagocytosis / Q000187:drug effects*; D013379:Substrate Specificity
chemical_list: D001643:Bridged Bicyclo Compounds; D004268:DNA-Binding Proteins; D004224:Diterpenes; C539619:Lsr2 protein, Mycobacterium tuberculosis