Structural basis for stereoselective dehydration and hydrogen-bonding catalysis by the SAM-dependent pericyclase LepI

Abstract

LepI is an S-adenosylmethionine (SAM)-dependent pericyclase that catalyses the formation of the 2-pyridone natural product leporin C. Biochemical characterization has shown that LepI can catalyse stereoselective dehydration to yield a reactive (E)-quinone methide that can undergo bifurcating intramolecular Diels-Alder (IMDA) and hetero-Diels-Alder (HDA) cyclizations from an ambimodal transition state, as well as a [3,3]-retro-Claisen rearrangement to recycle the IMDA product into leporin C. Here, we solve the X-ray crystal structures of SAM-bound LepI and in complex with a substrate analogue, the product leporin C, and a retro-Claisen reaction transition-state analogue to understand the structural basis for the multitude of reactions. Structural and mutational analysis reveals how nature evolves a classic methyltransferase active site into one that can serve as a dehydratase and a multifunctional pericyclase. Catalysis of both sets of reactions employs H133 and R295, two active-site residues that are not found in canonical methyltransferases. An alternative role of SAM, which is not found to be in direct contact with the substrate, is also proposed.

Fig. 1 |. Leporin C biosynthesis pathway highlighting LepI-catalyzed reaction cascade.

In the absence of LepI, spontaneous dehydration of alcohol 2 yields a (E/Z)-mixture of quinone methides (3/4), which nonenzymatically form Diels-Alder and hetero Diels-Alder adducts. The compounds used in structural study are highlighted by maroon boxes.

Fig. 2 |. LepI structure and the SAM binding site.

(a) The overall tertiary structure of LepI is shown in cartoon model, with the N-terminal dimerization domain and C-terminal catalytic domain colored in blue and red, respectively. Simulated-annealing omit map (grey mesh, contoured at 2.5 σ) indicates binding of SAM at the canonical SAM binding site. (b) Intimate LepI homodimer featured by an intertwined dimer interface. (c) Close-up view of SAM binding site. Hydrogen bond interactions are indicated with black dashes. (d) Next to SAM is a large substrate binding cavity and its entrance tunnel (shown together as green surface). The volume of cavity was calculated using POCASA.

Fig. 3 |. Crystal structure of LepI pseudo enzyme-substrate complex and enzyme-product complex.

Simulated-annealing omit maps are shown in black mesh and contoured at 3.0 σ. Hydrogen bond interactions are indicated with black dashed lines. (a) Crystal structure of LepI-SAM-1 (pseudo enzyme-substrate complex). Note that two rotamers of R295 and M45(B) are observed, and two conformations of diene (s-trans and s-cis) are modeled according to the electron density. Residues from monomer A are colored in salmon, whereas residues from monomer B are colored in light blue and labeled with B in parenthesis. (b) Superposition of LepI-SAM-1 complex (colored as in (a)) with unliganded LepI-SAM (residues are colored in dark green while SAM is colored in black). Substantial conformational changes are observed for F189, R295, R197, and M45(B). (c) Crystal structure of LepI-SAM-10. Residues and ligands are color coded as in (a). (d) Superposition of LepI-SAM-10 (residues are colored in white, SAM is colored in gray, ligand is colored in orange) with LepI-SAM-1 (color coded as in (a), note that ethylene glycol is not shown here for clarity).

Fig. 4. LepI Mutant activity.

(a) In vivo activity of LepI or mutants to synthesize leporin C (10) starting from the alcohol substrate 2. 2 is synthesized by upstream biosynthetic enzymes; for details of pathway see Ref. . (b) In vitro retro-Claisen rearrangement activity using 9 as the substrate. Asterisks indicate mutants with no measurable activity. Error bars show square deviation (s.d.) of three independent experiments (n = 3).

Fig. 5 |. Proposed catalytic mechanism of LepI.

Dashed straight lines indicate hydrogen bonds.