Characterization of the Rv3378c Gene Product, a New Diterpene Synthase for Producing Tuberculosinol and (13R,S)-Isotuberculosinol (Nosyberkol), from theMycobacterium tuberculosisH37Rv Genome

Nakano, Chiaki; Ootsuka, Takahiro; Takayama, Kazutoshi; Mitsui, Toshiaki; Sato, Tsutomu; Hoshino, Tsutomu

doi:10.1271/bbb.100570

Cited by 31 publications

(83 citation statements)

References 14 publications

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“…Rv3377c is a terpene cyclase, which acts on geranylgeranyl pyrophosphate (GGPP) to generate tuberculosinyl pyrophosphate (TbPP). Rv3378c was thought to be a phosphatase, which converts TbPP to free tuberculosinol (10,21). Extending current models (Fig.…”

Section: Resultsmentioning

confidence: 65%

“…2). The NMR signals of the diterpene moiety matched those of tuberculosinol (10,(19)(20)(21) except for the expected difference in the side-chain protons and carbons. The spectral data of the adenosine and adjacent atoms correspond closely to those of 1-prenyladenosine analogs (22)(23)(24).…”

Section: Resultsmentioning

confidence: 70%

“…3B). The concurrently performed biochemical studies described above identified the highly characteristic tuberculosinyl moiety as a component of 1-TbAd, and the Rv3377c-Rv3378c locus was known to encode enzymes needed for tuberculosinol and isotuberculosinol production (10,11,(18)(19)(20)(21). Sequencing identified spontaneous mutations in Rv3378c in both mutants (10,(18)(19)(20)(21).…”

Section: Resultsmentioning

confidence: 98%

“…Rv3378c has few orthologs in nature, and its biochemical function was not apparent from predictive folding algorithms. Based on in vitro studies, Rv3378c is currently thought to function as a TbPP pyrophosphatase that yields free tuberculosinol (10). Tuberculosinol coupled to beads blocks phagosomal acidification (21).…”

Section: Discussionmentioning

confidence: 99%

“…Previously, Rv3378c was thought to generate free tuberculosinol and isotuberculosinol (10)(11)(12). This discovery revises the enzymatic function of Rv3378c, which acts as a virulence factor to inhibit phagolysosome fusion (13).…”

Section: Significancementioning

confidence: 99%

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Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

Layre

Lee

Young

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.TbAd | terpenyl transferase W ith a mortality rate exceeding 1.5 million deaths annually, Mycobacterium tuberculosis remains one of the world's most important pathogens (1). M. tuberculosis succeeds as a pathogen because of productive infection of the endosomal network of phagocytes. Its residence within the phagosome protects it from immune responses during its decades long infection cycle. However, intracellular survival depends on active inhibition of pH-dependent killing mechanisms, which occurs for M. tuberculosis but not species with low disease-causing potential (2). Intracellular survival is also enhanced by an unusually hydrophobic and multilayered protective cell envelope. Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (3), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass and LipidDB databases (4, 5).To broadly compare the lipid profiles of virulent and avirulent mycobacteria, we took advantage of a recently validated metabolomics platform (4). This high performance liquid chromatography-mass spectrometry (HPLC-MS)...

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Section: Resultsmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

Layre

Lee

Young

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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Terpen‐Biosynthese: Modularitätsregeln

Oldfield

Lin

2011

Angewandte Chemie

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1150 www.angewandte.de EinleitungTerpene oder Isoprenoide bilden die vielfältigste Naturstoffklasse und kommen in fast allen Lebensformen vor, wo sie unzählige Aufgaben -von hauptsächlich strukturellen (Cholesterin in Zellmembranen) bis funktionalen (Carotinoide bei der Photosynthese; Retinal beim Sehvorgang; Chinone beim Elektronentransfer) -übernehmen. [1] Tatsächlich leiten sich alle -zumindest teilweise -von den C 5 -Substraten Dimethylallyldiphosphat (DMAPP, 1; Schema 1) und Isopentenyldiphosphat (IPP, 2) ab: Normalerweise wird zunächst DMAPP in einer 1'-4-oder "Kopf-Schwanz"-Kondensation mit einem oder mehreren IPP-Molekülen unter Bildung von Geranyldiphosphat (GPP, 3; C 10 ), Farnesyldiphosphat (FPP, 4; C 15 ) oder Geranylgeranyldiphosphat (GGPP, 5; C 20 ) verknüpft. [2] FPP und GGPP kçnnen dann in "Kopf-Kopf"-Anordnung (manchmal auch als Schwanz-Schwanz-Anordnung bezeichnet [4] ) kondensieren, [3] um beispielsweise Dehydrosqualen (DHS, 6), Squalen (7) oder Phytoen (8) zu bilden, die Vorläufer von Carotinoiden wie b-Carotin (9), Sterolen wie Cholesterin (10) und Hopanoiden wie Bakteriohopantetrol (11) -einige der ältesten und häufigsten Naturstoffe. [1] Isoprenoide kçnnen auch verwendet werden, um Proteine posttranslational zu modifizieren (wichtig für die Signalübertragung in der Zelle), oder sie kçnnen zu zahlreichen Terpen-Naturstoffen cyclisiert werden: zu C 10 -Monoterpenen wie Menthol (12), C 15 -Sequiterpenen wie Farnesen (13) und Artemisinin (14) und C 20 -Diterpenen, die z. B. in Gibberellinsäure (15) und Taxol (16) überführt werden. Durch Pflanzen wird DMAPP in einem Ausmaß von ungefähr 100 Megatonnen pro Jahr in Isopren (17) umgewandelt -eine Reaktion, die als potenzielle Quelle für "erneuerbare" Brennstoffe und andere Produkte gegenwärtig interessant ist. [5] Die DMAPP-und IPP-Vorstufen werden über zwei verschiedene Reaktionswege synthetisiert: den Mevalonat- [6] und den Methylerythritolphosphat(MEP)-Weg. [7] Der Mevalonat-Weg wird von den meisten Eukaryoten (einschließlich Menschen) sowie von Archaebakterien [8] genutzt, während der MEP-Weg in den meisten Eubakterien vorkommt. Es gibt natürlich Ausnahmen, z. B. verwendet das Bakterium Staphylococcus aureus den Mevalonat-Weg, während (eukaryotische) Malariaparasiten den MEP-Weg nutzen. [9] In Pflanzen kommen beide Biosynthesewege vor, [7] wobei der MEP-Weg üblicherweise in den Plastiden zu finden ist und der Mevalonat-Weg im Cytosol: Sterole (Triterpene) werden über Mevalonat synthetisiert, während Hemi-, Mono-und Diterpene sowie Carotinoide (Tetraterpene) über MEP aufgebaut werden. Im Folgenden erläutern wir neuere Erkenntnisse bezüglich Struktur und Funktion bei vielen Schlüsselenzymen der Isoprenoid-Biosynthese: den Kopf-Terpene bilden die umfangreichste Klasse niedermolekularer Naturstoffe auf der Erde. Hier werden neuere Entwicklungen bei der Aufklärung von Struktur und Funktion der an der Terpen-Biosynthese beteiligten Proteine zusammengefasst. Die Strukturen dieser Enzyme bestehen aus sechs Hauptbausteinen oder Modulen (a,b,g,d,e und z...

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Terpene Biosynthesis: Modularity Rules

2011

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Terpenes are the largest class of small molecule natural products on Earth, and the most abundant by mass. Here, we summarize recent developments in elucidating the structure and function of the proteins involved in their biosynthesis. There are 6 main building blocks or modules (α,β,γ,δ,ε and ζ) that make up the structures of these enzymes: the αα and αδ head-to-tail trans-prenyl transferases that produce trans-isoprenoid diphosphates from C5 precursors; the ε head-to-head prenyl transferases that convert these diphosphates into the tri-and tetra-terpene precursors of sterols, hopanoids and carotenoids; the βγ di- and tri-terpene synthases; the ζ head-to-tail cis-prenyl transferases that produce the cis-isoprenoid diphosphates involved in bacterial cell wall biosynthesis, and finally the α, αβ and αβγ terpene synthases that produce plant terpenes, with many of these modular enzymes having originated from ancestral α and β domain proteins. We also review progress in determining the structure and function of the two 4Fe-4S reductases involved in formation of the C5 diphosphates in many bacteria, where again, highly modular structures are found.

show abstract

Characterization of the Rv3378c Gene Product, a New Diterpene Synthase for Producing Tuberculosinol and (13R,S)-Isotuberculosinol (Nosyberkol), from theMycobacterium tuberculosisH37Rv Genome

Cited by 31 publications

References 14 publications

Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

Terpen‐Biosynthese: Modularitätsregeln

Terpene Biosynthesis: Modularity Rules

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