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

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.


Supplementary Figures
Supplementary Figure 1 | The selected enzymatic cyclopropanation strategies in natural product biosynthesis. According to the degree of dependence on SAM, these cyclopropanation strategies can be divided into three classes. Class I, a. formation of cyclopropane-containing terpenoids via inter-and intramolecular electrophilic addition; b. biosynthesis of the alkaloid cycloclavine through an α-ketoglutarate-dependent, non-heme iron oxygenase EasH-catalyzed oxidative rearrangement (three proposed mechanisms); and c. construction of cyclopropane-containing building blocks for nonribosomal peptides and hybrid nonribosomal peptide-polyketide compounds using halogenated carrier protein-linked intermediates as the substrates for S N 2-like cyclopropanation. Class II, formation of cyclopropane fatty acids by cyclopropane fatty acid/mycolic acid synthases that catalyze direct transfer of the reactive one-carbon species from SAM to double bonds involving a mechanism of carbocationic intermediates (or transition states). Class III, biosynthesis of 1-aminocyclopropane-1-carboxylate as a precursor to the plant hormone ethylene, and of the cyclopropane warhead of colibactin through a carbanion-induced intramolecular S N 2 reaction mechanism with elimination of methylthioadenosine.

Supplementary Figure 2 | Construction and verification of the Δc10Q mutant strain S. zelensis TG1405.
Verification was performed by PCR amplification using the genomic DNA from the mutant or wild type strain as the template. The PCR primers are labeled with their predicted sizes of the resulting products. [M+H] + c.  The result shows that the Ka value is 1.25×10 5 M -1 , indicating that there is a strong protein-protein interaction between Swoo_2002 and C10Q.    HemN Streptomyces corchorusii strain DSM 40340, and NosN from the biosynthetic gene cluster of nosiheptide. The alignment was carried out using CLUSTAL Omega (1.2.1). The conserved motif CxxxCxxC for binding [4Fe-4S] cluster is marked in green, the conserved glutamine and arginine residues for binding SAM 1 selected for mutation are marked in cyan, and the conserved tyrosine residue for binding SAM 2 selected for mutation is marked in yellow.
Swoo_2002 C57A + C10Q Swoo_2002 + C10Q   In pathway a, the carbon-centered radical at C-12 in 9 abstracts a solvent-exchangeable proton to produce the intermediate 8.
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. Pathway b is an alternative proposal for the conversion from 9 to 7, that is, the carbon-centered radical at C-12 in 9 triggers the removal of a proton at C-11 by an unknown base with elimination of SAH, and then the formed allylic radical is quenched at the C-11M position. However, pathway b is in conflict with our labeling experiments result that the D atom in the produced D-7 is either located in the C-12 or C-11M position but not merely in C-11M position when using D 2 O instead of H 2 O in the enzymatic assay.

Supplementary Tables
Supplementary The BGC of yatakemycin has been previously cloned in our group 2 . The gilvusmycin has been connected with two potential bacterial strains 2 . Eight of cryptic BGCs have been assigned potential producer of CC-1065 3 .