Identification of the First Bacterial Monoterpene Cyclase, a 1,8‐Cineole Synthase, that Catalyzes the Direct Conversion of Geranyl Diphosphate

Nakano, Chiaki; Kim, Hyo-Kyoung; Ohnishi, Yasuo

doi:10.1002/cbic.201100330

Cited by 57 publications

(53 citation statements)

References 22 publications

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“…For example, cluster 3 contains pentalenene synthases, an avermitilol synthase, an acyclic terpene synthase that produces nerolidol and linalool, and a monoterpene cyclase that produces 1,8-cineole (13,14,31,32). The product diversity observed in this cluster suggests that many of the unknown terpene synthases in this cluster that are annotated as pentalenene synthases may be incorrect.…”

Section: Resultsmentioning

confidence: 99%

“…In 2011, Hu et al reported that two other terpene synthases in this organism are involved in biosynthesis of T-muurolol and δ-cadinene (10). In the same year, Nakano et al reported the characterization of two additional terpene synthases, a monoterpene cyclase that produced 1,8-cineole and a promiscuous acyclic terpene synthase that produced linalool and nerolidol (13,14). While this manuscript was in the final stages of preparation, the function of an additional terpene synthase, which is a protein variant of the subject of this investigation, was reported to synthesize a linear triquinane of undefined stereochemistry (15).…”

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confidence: 91%

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Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Chow

Tian

Ramamoorthy

et al. 2015

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

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Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.T erpenoid compounds form a large group of natural products synthesized by many species of plants, fungi, and bacteria. To date, more than 70,000 of these compounds have been identified, comprising ∼25% of all of the known natural products (Dictionary of Natural Products database, dnp.chemnetbase.com/intro/ index.jsp). Terpenoids and molecules containing terpenoid fragments are produced from two basic five-carbon building blocks: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (1, 2). IPP and DMAPP are then converted into a series of terpenoid diphosphate esters of increasing chain lengths by chain length selective polyprenyl diphosphate synthases to give geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate (FPP, C 15 ), geranylgeranyl diphosphate (GGPP, C 20 ), and higher five-carbon homologs (3). These molecules are substrates for terpene synthases, which catalyze hydroxylation, elimination, cyclization, and rearrangement reactions to produce much of the structural diversity of terpenoid molecules found in nature (4, 5).The two main classes of terpene synthases (class I and class II) are distinguished from one another by the mechanisms they use to initiate carbocationic cyclization and rearrangement reactions and by their respective folds (6, 7). Class I terpene synthases use active site Mg 2+ ions to bind and activate the diphosphate moieties of their substrates as leaving groups to generate highly reactive allylic carbocations. In those terpene synthases that catalyze cyclization reactions, the carbocations alkylate distal double bonds in the hydrocarbon chain. The initial cycliza...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 91%

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Chow

Tian

Ramamoorthy

et al. 2015

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

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“…The terpenes that were produced by heterologous expression all accumulated in the mycelium, except for 1,8-cineole, which was generated by expression of sclav_p0982 using the vector pKU1021gps. SCLAV_p0982 has previously been shown directly by in vitro incubation to catalyze the cyclization of GPP to 1,8-cineole (47). Expression of sclav_p0982 in the heterologous Streptomyces host established that the expressed terpene synthase generates not only 1,8-cineole but also, β-pinene and camphene.…”

Section: Heterologous Expression Of Genes Encoding Bacterial Terpenementioning

confidence: 97%

Terpene synthases are widely distributed in bacteria

Yamada

Kuzuyama

Komatsu

et al. 2014

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

442

366

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Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.terpene synthase | bacteria | heterologous expression

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“…38 This result, together with several genome mining studies for SC homologs by our group [12][13][14][15] and others, [2][3][4][5][6][7][8][9][10][11] indicates that terpenoids are significantly more widely distributed in nature than was previously appreciated, being widespread among not only eukaryotes, but also bacteria.…”

Section: Discussionmentioning

confidence: 61%

“…Recently, we reported the identification and characterization of five bacterial single-domain SC homologs: the ( þ )-caryolan-1-ol synthase GcoA 12 in Streptomyces griseus, the 1,8-cineole synthase CnsA 13 and linalool/nerolidol synthase LnsA 14 in Streptomyces clavuligerus, and the ( À)-germacradien-4-ol synthase SC1 15 and ( À)-epi-a-bisabolol synthase SC2 15 in Streptomyces citricolor. CnsA is the first bacterial monoterpene synthase known to catalyze the direct conversion of geranyl diphosphate, 13 whereas LnsA is the first bacterial acyclic terpene synthase that shows significant amino-acid sequence similarity to bacterial SCs. 14 These studies and the presence of a large number of terpene cyclase homologs in bacteria indicate that terpenoids are likely to be significantly more widely distributed in nature than previously appreciated.…”

Section: Introductionmentioning

confidence: 99%

Identification of the SGR6065 gene product as a sesquiterpene cyclase involved in (+)-epicubenol biosynthesis in Streptomyces griseus

et al. 2012

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Recent bacterial genome sequencing projects have shown the presence of many putative sesquiterpene cyclase (SC) genes, especially in the Gram-positive, filamentous bacterial genus Streptomyces. We describe here the characterization of a SC gene (SGR6065, named gecA) from Streptomyces griseus. Overexpression of gecA in Streptomyces lividans produced a sesquiterpene, which was isolated and determined to be ( þ )-epicubenol using spectroscopic analyses. The N-terminal histidine-tagged GecA protein was produced in Escherichia coli. Incubation of the recombinant GecA protein with farnesyl diphosphate (FPP) yielded ( þ )-epicubenol as the major product. The K m value for FPP and the k cat value for ( þ )-epicubenol formation were calculated to be 254 ± 7.1 nM and 0.026 ± 0.001 s À1 , respectively. The k cat /K m value (0.10 s À1 lM À1 ) was broadly comparable to those reported for known bacterial SCs. ( þ )-Epicubenol was detected in the crude cell lysate of wild-type S. griseus, but not in a gecA-knockout mutant, indicating that GecA is a genuine ( þ )-epicubenol synthase. Although ( þ )-epicubenol synthases have been previously purified and characterized from the liverwort Heteroscyphus planus and Streptomyces sp. LL-B7, no ( þ )-epicubenol synthase gene has been cloned to date. The gecA gene is thus the first example of an ( þ )-epicubenol synthase-encoding gene. ( þ )-Epicubenol production was not controlled by the microbial hormone A-factor that induces morphological differentiation and production of several secondary metabolites in S. griseus.

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Identification of the First Bacterial Monoterpene Cyclase, a 1,8‐Cineole Synthase, that Catalyzes the Direct Conversion of Geranyl Diphosphate

Cited by 57 publications

References 22 publications

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Terpene synthases are widely distributed in bacteria

Identification of the SGR6065 gene product as a sesquiterpene cyclase involved in (+)-epicubenol biosynthesis in Streptomyces griseus

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