Expression and mechanistic analysis of a germacradienol synthase from
            <i>Streptomyces coelicolor</i>
            implicated in geosmin biosynthesis

Cane, David E.; Watt, RM

doi:10.1073/pnas.0337625100

Cited by 132 publications

(153 citation statements)

References 30 publications

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“…1A). Terpene synthases with similar functions sometimes group in the same clusters, as illustrated by epi-isozizaene synthases (cluster 4), 2-methylisoborneol synthases (cluster 7), aristolochene synthases (cluster 13), and geosmin synthases (cluster 8) (27)(28)(29)(30). (See Fig.…”

Section: Resultsmentioning

confidence: 99%

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%

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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“…3). In contrast to these eukaryotic isoprenoid cyclases, only limited studies on prokaryotic isoprenoid cyclases have been reported so far [10,62]. Therefore, studies were conducted to investigate the biosynthetic genes and enzymes of isoprenoid antibiotics produced by actinomycetes, particularly focusing on the cloning and characterization of the genes of the mevalonate pathway and the genes of isoprenoid cyclase.…”

Section: Biosynthesis Of Isoprenoidsmentioning

confidence: 99%

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Dairi

2005

J Antibiot

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Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMGCoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.Keywords isoprenoid, mevalonate pathway, biosynthesis, cyclase, Actinomycetes IntroductionActinomycete strains usually produce a number of secondary metabolites that often possess pharmaceutical activities. Therefore, for a long time, the strains have been used for many screenings to find novel, medically useful compounds. Consequently, more than 60 percent of the known antibiotics, including not only antibacterial antibiotics but also bioactive microbial compounds, have been reported to be produced by actinomycetes [1]. Presently, such pharmaceutically important, novel compounds can be found in the culture broth of actinomycete strains by screening rare actinomycetes, developing new screening strategies, employing sensitive assay methods for the detection of low concentrations of antibiotics, etc. However, sometimes, "semi-new antibiotics"-derivatives of known antibiotics-were isolated; discovering novel antibiotics every year became very difficult.In the past two decades, recombinant DNA technology has come into play in this field. Gene clusters encoding many natural products have been cloned and characterized. Moreover, whole-genome sequencing has uncovered hundreds of candidates for secondary metabolic pathways. Among them, the biosynthetic machinery of nonribosomal peptide and polyketide natural products has been extensively investigated [2ϳ7]. Recent progress in the application of combinatorial biosynthesis methods to these bi...

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“…Dr Rory Watt (now at the University of Hong Kong) then expressed the Cyc2 protein and established that it catalyzed cyclization of FPP to the monocyclic sesquiterpene alcohol (4S,7R)-germacradienol (17), an activity that he also showed was due exclusively to the N-terminal half of the protein (Figure 12). 92 This discovery, however interesting, might have remained only a curiosity, had not Keith Chater, Greg Challis and their collaborators at the John Innes, using their newly developed method for generating directed in-frame gene knockouts in Streptomyces, discovered simultaneously that the same 2.3-kb gene is essential to the biosynthesis of the well-known odoriferous compound geosmin (18), a volatile degraded sesquiterpene produced by all Streptomyces that is responsible for the universally recognized odor of moist soil. 93 Two graduate students in Editorial my laboratory, Xiaofei He (now at Shanghai Jiaotong University) and Jiaoyang Jiang (now an Assistant Professor at the School of Pharmacy at the University of Wisconsin), then made the unexpected discovery that germacradienol synthase is in fact a bifunctional geosmin synthase enzyme that converts FPP all the way to geosmin, with the C-terminal domain catalyzing an unprecedented cyclization-fragmentation of the initially formed germacradienol (Figure 12).…”

Section: Enzymology and Molecular Genetics Of Natural Product Biosyntmentioning

confidence: 99%

Nature as organic chemist

Cane

2016

J Antibiot

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Figure 15 Stereospecific reduction of (2R)-2-methyl-3-ketopentanoyl-ACP by recombinant ketoreductase (KR) domains: EryKR6, from module 6 of the 6dEB synthase (DEBS); TylKR1, from module 1 of the tylactone synthase; EryKR1, from DEBS module 1; RifKR7, from module 7 of the rifamycin PKS. The (2R)-2-methyl-3-ketopentanoyl-ACP substrate is generated in situ by a reconstituted mixture of dissected PKS domains, Ery[KS6][AT6] (ketosynthase-acyl transferase didomain) and EryACP6 are both from DEBS module 6. Editorial

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Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis

Cited by 132 publications

References 30 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

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Nature as organic chemist

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