Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.