A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis - PubMed (original) (raw)

A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis

Wen-Bing Jin et al. Nat Commun. 2018.

Abstract

Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-L-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp2-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular SN2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Chemical structures of the spirocyclopropylcyclohexadienone family of natural products. This family of natural products includes CC-1065 (1), gilvusmycin (2), yatakemycin (YTM, 3), duocarmycin SA (4), and duocarmycin A (5)

Fig. 2

Fig. 2

Characterization of a two-component cyclopropanase system. a–h HPLC analysis of the relevant metabolites (UV at 374 nm). a Standard of 1.b Standard of 6. c Wild type Streptomyces zelensis NRRL 11183. d The Δ_c10P_ mutant. e The Δ_c10Q_ mutant. f The Δ_c10Q_ mutant complementated with the c10Q gene. g The Δ_c10P_ mutant complementated with the Swoo_2002 gene. h The Δ_c10P_ mutant complementated with the c10P gene. i Structures of 6 and 7. j–q HPLC analysis of enzymatic products. j–m are the control reactions without C10P, C10Q, Na2S2O4, and SAM, respectively. n Standard of 1. o The complete reaction for C10P and C10Q. p The complete reaction for Swoo_2002 and C10Q. q The reaction for Swoo_2002 and C10Q H138A. All the in vitro enzymatic activity assays were performed in an anaerobic glove box with less than 1 ppm of O2. Reactions were conducted in Tris•HCl buffer (Tris 50 mM, NaCl 100 mM, glycerol 10%, pH 8.0) with the following composition: 1 μM reconstituted Swoo_2002, 2 μM C10Q, 10 μM substrate 6, 1 mM SAM, 5 mM DTT, 5 mM MgCl2, 5 mM Na2S2O4 and 7% DMSO. The reactions were incubated at 28 °C for 12 h

Fig. 3

Fig. 3

Analysis of reaction products from the cyclopropanation process. a–e HPLC analysis of the enzymatic products (UV at 260 nm). a Standard of 5′-dA. b Standard of SAH. c The control reaction using boiling-inactivated enzymes. d, e are the concentrated products from a large-scale enzymatic assay terminated at 4 h and subjected to evaporation and lyophilization, respectively. f Time-course analysis of the concentration changes of 1, 6, 7, 5′-dA, and SAH from the cyclopropanation process. g, h are the HR-MS analyses of the intermediate 8 from the reactions using SAM and CD3-SAM, respectively. i MS/MS analysis of 8 using SAM. j The chemical structure and calculated molecular weight of the intermediate 8

Fig. 4

Fig. 4

Isotope labeling investigations into the cyclopropanation process. a–f HR-MS analysis of the products from reactions using SAM or CD3-SAM. a, c, e are the products of 5′-dA, 1, and 7, respectively, from the assay using SAM. b, d, f are the products of D-5′-dA, D 2 -1, and D 2 -7, respectively, from the assay using CD3-SAM. g-j HR-MS analysis of the products from reactions using H2O or D2O. g, i are the products of 1 and 7, respectively, from the assay using H2O. h, j are the products of D-1 and D-7, respectively, from the assay using D2O. k and l are the NMR analyses of the enzymatic product 7 from H2O (7, 1H-NMR) and D2O (D-7, 2H-NMR)

Fig. 5

Fig. 5

Proposed mechanism of the cyclopropanation process. Upon dithionite reduction, the [4Fe-4S]2+ from the HemN-like radical SAM enzyme is converted to [4Fe-4S]+, which triggers the reductive cleavage of the first molecule of SAM1 to yield a highly reactive dAdo radical. Then, the dAdo radical abstracts a hydrogen atom from the methyl group of the second molecule of SAM2. A SAM methylene radical is thus produced and then adds to the C-11 position of the substrate 6 to generate a radical intermediate 9. The carbon-centered radical at C-12 in 9 abstracts a solvent-exchangeable proton to produce the intermediate 8. Subsequently, the His-138 residue from C10Q likely functions as a critical base and deprotonates the phenolic hydroxyl group (C-6) of 8, which induces the intramolecular SN2 cyclopropanation to yield 1 with elimination of SAH as a co-product. On the other hand, the intermediate 8 may be non-enzymatically converted to the intermediate 10 containing an exocyclic double bond via release of SAH, followed by rapid and thermodynamic driving isomerization to give a methylated off-pathway compound 7

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