Characterization of the Rv3378c Gene Product, a New Diterpene Synthase for Producing Tuberculosinol and (13R, S)-Isotuberculosinol (Nosyberkol), from the Mycobacterium tuberculosis H37Rv Genome

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2011, Vol.75 (1), p.75-81
• Main Authors: NAKANO, Chiaki, OOTSUKA, Takahiro, TAKAYAMA, Kazutoshi, MITSUI, Toshiaki, SATO, Tsutomu, HOSHINO, Tsutomu
• Format: Article
• Language: English
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - isolation & purification; Alkyl and Aryl Transferases - metabolism; Amino Acid Motifs; Biological and medical sciences; Catalytic Domain; Chromatography, High Pressure Liquid; diterpene; Diterpenes - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Genome, Bacterial - genetics; isotuberculosinol; Kinetics; Magnetic Resonance Spectroscopy; Metals - pharmacology; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; nosyberkol; Protein Multimerization; Protein Structure, Quaternary; tuberculosinol
• ISSN: 0916-8451; 1347-6947; 1347-6947
• DOI: 10.1271/bbb.100570

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of 1 H- and 13 C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.