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Characterization and inhibition of a class II diterpene cyclase from Mycobacterium tuberculosis: implications for tuberculosis.

Mann FM, Prisic S, Hu H, Xu M, Coates RM, Peters RJ - J. Biol. Chem. (2009)

Bottom Line: Chem.Soc., in press).Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Iowa State University, Ames, Iowa 50011, USA.

ABSTRACT
Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.

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Relative MBP-MtHPS activity with various divalent metal ion cofactors. Black bars, 0.1 mm; gray bars, 1 mm; white bars, 10 mm.
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Figure 4: Relative MBP-MtHPS activity with various divalent metal ion cofactors. Black bars, 0.1 mm; gray bars, 1 mm; white bars, 10 mm.

Mentions: As with other characterized class II diterpene cyclases (14, 17–19), it was found in the initial characterization report that MtHPS requires Mg2+ as an enzymatic cofactor (7). After optimization of the assay for kinetic measurements, the ability of a variety of divalent cations to support MtHPS activity at various concentrations (0.1, 1, and 10 mm) was investigated. This included Mg2+, Co2+, Cu2+, Fe2+, Mn2+, Ca2+, and Zn2+, although because of interference with the secondary enzyme in our coupled assay (phosphatase), we were unable to measure kinetic rates with Mn2+ and Zn2+. Of the remaining divalent cations, MtHPS reacted most efficiently in the presence of low levels of Mg2+ (Fig. 4).


Characterization and inhibition of a class II diterpene cyclase from Mycobacterium tuberculosis: implications for tuberculosis.

Mann FM, Prisic S, Hu H, Xu M, Coates RM, Peters RJ - J. Biol. Chem. (2009)

Relative MBP-MtHPS activity with various divalent metal ion cofactors. Black bars, 0.1 mm; gray bars, 1 mm; white bars, 10 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749132&req=5

Figure 4: Relative MBP-MtHPS activity with various divalent metal ion cofactors. Black bars, 0.1 mm; gray bars, 1 mm; white bars, 10 mm.
Mentions: As with other characterized class II diterpene cyclases (14, 17–19), it was found in the initial characterization report that MtHPS requires Mg2+ as an enzymatic cofactor (7). After optimization of the assay for kinetic measurements, the ability of a variety of divalent cations to support MtHPS activity at various concentrations (0.1, 1, and 10 mm) was investigated. This included Mg2+, Co2+, Cu2+, Fe2+, Mn2+, Ca2+, and Zn2+, although because of interference with the secondary enzyme in our coupled assay (phosphatase), we were unable to measure kinetic rates with Mn2+ and Zn2+. Of the remaining divalent cations, MtHPS reacted most efficiently in the presence of low levels of Mg2+ (Fig. 4).

Bottom Line: Chem.Soc., in press).Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Iowa State University, Ames, Iowa 50011, USA.

ABSTRACT
Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.

Show MeSH
Related in: MedlinePlus