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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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Reaction catalyzed by HPS and subsequent production of edaxadiene (4). Shown is the acid-catalyzed protonation-initiated bicyclization of GGPP (1) to a copalyl diphosphate carbocation intermediate (2), the subsequent rearrangement via a series of alternating 1,2-hydride and methyl migrations to form the HPP (3a) product after terminating deprotonation, and the following separate additional cyclization of 3a to edaxadiene (4) catalyzed by Rv3378c/M. tuberculosis edaxadiene synthase (MtEDS).
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Figure 1: Reaction catalyzed by HPS and subsequent production of edaxadiene (4). Shown is the acid-catalyzed protonation-initiated bicyclization of GGPP (1) to a copalyl diphosphate carbocation intermediate (2), the subsequent rearrangement via a series of alternating 1,2-hydride and methyl migrations to form the HPP (3a) product after terminating deprotonation, and the following separate additional cyclization of 3a to edaxadiene (4) catalyzed by Rv3378c/M. tuberculosis edaxadiene synthase (MtEDS).

Mentions: A genetic screen focused on primary effects very early in the infection process strongly implicated the product of a five-gene isoprenoid biosynthetic operon (6). In particular, inactivating transposon insertion in the two unique (presumably non-redundant) genes in the operon resulted in mutant M. tuberculosis unable to fully block phagosomal maturation. Closely following work demonstrated that the first of these, Rv3377c, encoded a class II diterpene cyclase that catalyzed bicyclization and rearrangement of (E,E,E)-geranylgeranyl diphosphate (GGPP4; 1a) to halimadienyl diphosphate (HPP; 3a) (Fig. 1) (7). The corresponding enzyme has been termed halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Recently, we have reported that the second implicated gene, Rv3378c, encodes a subsequently acting class I diterpene cyclase that further cyclizes HPP (3a) to the novel tricyclic diterpene edaxadiene (4), which directly inhibits phagosomal maturation in vitro (8), consistent with the results of the previously reported genetic screen (6). Edaxadiene (4) then presumably contributes, at least at an early stage in the infection process, to the phagosomal arrest that provides M. tuberculosis with its host cell/compartment, with HPS catalyzing the committed step in its biosynthesis.


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)

Reaction catalyzed by HPS and subsequent production of edaxadiene (4). Shown is the acid-catalyzed protonation-initiated bicyclization of GGPP (1) to a copalyl diphosphate carbocation intermediate (2), the subsequent rearrangement via a series of alternating 1,2-hydride and methyl migrations to form the HPP (3a) product after terminating deprotonation, and the following separate additional cyclization of 3a to edaxadiene (4) catalyzed by Rv3378c/M. tuberculosis edaxadiene synthase (MtEDS).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Reaction catalyzed by HPS and subsequent production of edaxadiene (4). Shown is the acid-catalyzed protonation-initiated bicyclization of GGPP (1) to a copalyl diphosphate carbocation intermediate (2), the subsequent rearrangement via a series of alternating 1,2-hydride and methyl migrations to form the HPP (3a) product after terminating deprotonation, and the following separate additional cyclization of 3a to edaxadiene (4) catalyzed by Rv3378c/M. tuberculosis edaxadiene synthase (MtEDS).
Mentions: A genetic screen focused on primary effects very early in the infection process strongly implicated the product of a five-gene isoprenoid biosynthetic operon (6). In particular, inactivating transposon insertion in the two unique (presumably non-redundant) genes in the operon resulted in mutant M. tuberculosis unable to fully block phagosomal maturation. Closely following work demonstrated that the first of these, Rv3377c, encoded a class II diterpene cyclase that catalyzed bicyclization and rearrangement of (E,E,E)-geranylgeranyl diphosphate (GGPP4; 1a) to halimadienyl diphosphate (HPP; 3a) (Fig. 1) (7). The corresponding enzyme has been termed halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Recently, we have reported that the second implicated gene, Rv3378c, encodes a subsequently acting class I diterpene cyclase that further cyclizes HPP (3a) to the novel tricyclic diterpene edaxadiene (4), which directly inhibits phagosomal maturation in vitro (8), consistent with the results of the previously reported genetic screen (6). Edaxadiene (4) then presumably contributes, at least at an early stage in the infection process, to the phagosomal arrest that provides M. tuberculosis with its host cell/compartment, with HPS catalyzing the committed step in its biosynthesis.

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