Generation of Complexity in Fungal Terpene Biosynthesis: Discovery of a Multifunctional Cytochrome P450 in the Fumagillin Pathway

Lin, Hsiao‐Ching; Tsunematsu, Yuta; Dhingra, Sourabh; Xu, Wei; Fukutomi, Manami; Chooi, Yit‐Heng; Cane, David E.; Calvo, Ana M.; Watanabe, Kenji; Tang, Yi

doi:10.1021/ja500881e

Cited by 93 publications

(90 citation statements)

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“…In the actual intermediate Fe 2+ -O 2 -pentalenolactone F complex, it is of course likely that water W1 is either further shifted by the oxygen atom of the bound O 2 , or displaced altogether, with the remainder of the proton relay network remaining essentially intact. 19 Notably, the well-characterized proton relay networks in two other well-studied P450s, Pseudomonas putida cytochrome P450 cam , which catalyzes the C5 exo-hydroxylation of camphor, 18,20 and Saccharopolyspora erythraea P450eryF, 3b,21 which catalyzes the C6-hydroxlation of 6-deoxyerythronolide B, the parent macrolide aglycone of erythromycin A, are each anchored by homologous glutamate residues, P450 cam -E366 and P450eryF-E360, respectively, which occupy identical sites in the corresponding L-Helix of each protein. 20,21 Indeed, this glutamate is nearly universally conserved in all P450s, being found in >90% of the top 100 BLAST matches to the PntM sequence.…”

Section: Resultsmentioning

confidence: 99%

“…Although the conserved glutamates do not appear to have a continuous H-bonded link with bulk water at the protein surface, it has been proposed that the requisite proton delivery may involve concerted dynamic fluctuations of amino acid chains and bound waters. 3b,22 …”

Section: Resultsmentioning

confidence: 99%

“…1 Such reactions are catalyzed by both catabolic P450s, whose primary biological function is degradation of a broad range of foreign organic compounds 2a or mobilization of environmental organic chemicals as sources of energy and carbon, 2b as well as by metabolic P450s that catalyze substrate-specific reactions as part of multistep biosynthetic pathways. 3 …”

Section: Introductionmentioning

confidence: 99%

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The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

2016

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The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologues being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologues and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3re of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-•Fe3+-OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1si which is flanked on each neighboring carbon by syn-axial substituents.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

2016

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“…The explosion of newly sequenced microbial genomes has facilitated genome-mining efforts to discover new terpenoid metabolites. This process typically involves expressing the genes in the biosynthesis clusters and determining the function of each enzyme (40)(41)(42). This process is a nontrivial endeavor, and it is desirable to have some indication of the potential novelty of a metabolite when selecting which biosynthesis clusters to study.…”

Section: Discussionmentioning

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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“…This can be achieved either by the native fungal producer or more commonly, in the case of low-yield or genetically intractable strains, by engineered, recombinant systems. Therefore, there is a need to identify and characterize the natural products biosynthetic enzymes and pathways for the development of such heterologous production systems, facilitating access to these chemically diverse scaffolds (Ajikumar et al, 2010; Boettger and Hertweck, 2013; Lin et al, 2013; Lin et al, 2014; Paddon et al, 2013; Spakowicz and Strobel, 2015; Tsunematsu et al, 2013; Wawrzyn et al, 2012a; Wiemann et al, 2013; Wiemann and Keller, 2014; Yaegashi et al, 2014). …”

Section: Introductionmentioning

confidence: 99%

Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway

et al. 2016

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Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated the a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products.

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Generation of Complexity in Fungal Terpene Biosynthesis: Discovery of a Multifunctional Cytochrome P450 in the Fumagillin Pathway

Cited by 93 publications

References 53 publications

The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

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

Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway

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