Catalytic mechanism and endo-to- exo selectivity reversion of an octalin-forming natural Diels-Alderase

Abstract

We have previously reported the identification of CghA, a proposed Diels-Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA-product complex at a resolution of 2.0 Å. Our result provides the second structural determination of eukaryotic Diels-Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.

Extended Data Fig. 1 |. Classification of DAases by the mechanism of product release from the NRPS.

a, Proposed two distinct mechanisms of substrate release from the NrPS. One type of the embedded terminal reductase domain of an NrPS catalyses reduction and Knoevenagel condensation of the thioester intermediate to give a pyrrolin-2-one product via a rote A. Another type catalyses a Dieckmann condensation of the intermediate to give a pyrrolidine-2,4-dione product via a rote B. b, Phylogenetic tree of the lipocalin-type DAase homologs. Phylogenetic analysis of the amino acid sequences of the representative homologs found in the protein database revealed that they could be divided clearly into two groups based on the types of tetramic acid moieties described above that the corresponding products bear. Pink represents enzymes predicted to catalyse Knoevenagel condensation to release the pyrrolin-2-one-type (type A) product. Purple represents enzymes predicted to catalyse Dieckmann condensation to release the pyrrolidine-2,4-dione-type (type B) product. The length of the black bar is scaled to represent the evolutionary distance of 0.1 amino acid substitution per site. The enzyme names are abbreviated for the ease of representation.

Extended Data Fig. 2 |. Electron density of the bound ligand in the CghA co-crystal structure.

The Fo–Fc map contoured at 3.0σ represented as thin blue mesh is shown to indicate the electron density of Sch210972 bound within the active site pocket of CghA. Colouring scheme for the ligand is the same as in Fig. 2a.

Extended Data Fig. 3 |. Activity assays on the wild-type CghA and its mutants to examine the roles of the active-site residues.

The in vitro analyses were performed using the substrate analogue 8 for the formation of the endo 9 and exo 10 adducts. Detailed reaction conditions are described in Methods unless otherwise specified below. a, hPLC profiles of the reaction mixtures containing 8 as a substrate and (i) the S65N mutant; (ii) the N82A mutant; (iii) the h94A mutant; (iv) the K352A mutant; (v) the N364A mutant; (vi) the wild-type CghA; (vii) buffer, as a background reaction showing the conversion of 8 to 9 and 10 after 10 minutes of incubation; (viii) the authentic reference of 9; and (ix) the authentic reference of 10. The reactions were monitored at 288 nm. N-Boc-L-tryptophan methyl ester was used as an internal standard (IS) throughout the study. See Methods for details. Peaks denoted by asterisks are impurities from the chemical synthesis of 8. b, hydrogen-bonding interaction between the bound ligand P-1 and the S65 and N82 side chain groups. Two water molecules also link the S65 and N82 side chain groups through hydrogen bonds.

Extended Data Fig. 4 |. Background conversion of the substrate analogue 8.

Spontaneous transformation of the substrate analogue 8 into the endo 9 and exo 10 adducts occurs almost entirely in the hPLC solvent supplemented with formic acid. 8 (150 μM) was incubated for 0 (labelled “buffer 0 min” in the plot), 10 (buffer 10 min) and 20 min (buffer 20 min) at 25 °C without CghA in the reaction buffer (100 mM potassium phosphate, 100 mM NaCl, ph 8.4) in a total reaction volume of 25 μl. After incubation, the reaction was quenched with 25 μl of MeOh and then centrifuged for removal of debris. Subsequently, the sample was subjected to LC–MS analysis. For the control experiment “buffer 10 min + CghA 10 min”, 8 (150 μM) was incubated for 10 min at 25 °C without CghA in the reaction buffer to allow the spontaneous transformation of 8. Subsequently, 0.1 μM of the wild-type CghA was added to the reaction mixture and the mixture was incubated for another 10 min at 25 °C in a total reaction volume of 25 μl to allow CghA to react on the remaining 8. For other control experiments “CghA 10 min”, “CghA + 10 10 min” and “CghA + 9”, 150 μM of 8, 10 and 9 was added to the reaction mixture, respectively. These mixtures were incubated for 10 min at 25 °C with CghA (0.1 μM wild-type CghA) under the same conditions and analysed as described above.

Extended Data Fig. 5 |. Total turnover rates of the wild-type CghA and its mutants designed for stereospecificity conversion.

The combined apparent turnover rates kcat(app) for the formation of 9 and 10 from 8 by the CghA mutants that were designed to examine the role of octalin-packing residues in determining the diastereoselectivity of the adducts formed are presented. The centre value is the mean of triplicate measurements with each data point shown as an open circle and the error bar representing the standard deviation. See Methods and Supplementary Note 5 for details.

Extended Data Fig. 6 |. Activity assays on CghA and its mutants to examine endo-to-exo stereospecificity conversion.

The in vitro analyses were performed using the substrate analogue 8 as the substrate. Detailed reaction conditions are described in Methods unless otherwise specified below. The reactions were monitored at 288 nm. N-Boc-L-tryptophan methyl ester was used as an internal standard (IS) throughout the study. See Methods for details. Peaks denoted by asterisks are impurities from the chemical synthesis of 8. a, hPLC profiles of the reaction mixtures containing 8 as a substrate and (i) buffer only as a background reaction; (ii) the wild-type CghA; (iii) the A242S mutant; (iv) the M257V mutant; (v) the V391L mutant; (vi) the A242S/M257V mutant; (vii) the A242S/M257V/V391L mutant; (viii) the authentic reference of 9; and (ix) the authentic reference of 10. b, hPLC profiles of the reaction mixtures containing 8 as a substrate and (i) buffer, as a background reaction; (ii) the wild-type CghA; (iii) the A242S/M257V/V391L mutant; (iv) the A242N/M257V/V391L mutant; (v) the authentic reference of 9; and (vi) the authentic reference of 10.

Extended Data Fig. 7 |. The kinetic assay to examine product inhibition of CghA.

In vitro analyses of CghA were performed with 8 as the substrate in the presence of an increasing concentration of the reaction product (the endo adduct 9) to examine the possible product inhibition of CghA. The activity at each endo product concentration is given relative to the activity in the absence of the product. The centre value is the mean of triplicate measurements with the error bar representing the standard deviation. See Methods for details.

Fig. 1 |. Octalin-containing fungal secondary metabolites and proposed involvement of Diels–Alder reactions in their biosynthesis.

a, Sch210972 (1), equisetin (2), myceliothermophin E (3). b, The proposed biosynthesis of 1. The hybrid polyketide synthase (PKS)–nonribosomal peptide synthetase (NrPS) CghG and the stand-alone enoyl reductase (Er) CghC together form the linear intermediate. The terminal reductase (r) domain of CghG is proposed to catalyse the tetramate moiety-forming Dieckmann-type condensation. CghA is thought to catalyse the cycloaddition reaction. KS, ketosynthase; MAT, malonyl-CoA acyltransferase; Dh, dehydratase; MT, methyltransferase; Kr, ketoreductase; ACP, acyl carrier protein; C, condensation; A, adenylation; T, thiolation; SAM, S-adenosyl-l-methionine.

Fig. 2 |. Crystal structure of CghA in complex with 1.

a, Overall structure of CghA and the bound product 1. b, Stereo view of the active site. Carbon atoms of the protein side chains and 1 are in green and yellow, respectively. Oxygen, nitrogen and sulfur atoms are in red, blue and gold, respectively. Dashed lines represent hydrogen bonds. c, Comparison of the CghA (green)–endo adduct (yellow) complex and a modelled A242S/M257V/V391L mutant (magenta)–exo adduct (cyan) complex. d, Conformational differences between the apo (purple) and complex (green) structures at W183, W235 and K352.

Fig. 3 |. In vitro assays of the CghA mutants.

a, The expected endo 9 and exo 10 adducts formed from the substrate analogue 8 by the spontaneous or enzyme-catalysed Diels–Alder reaction. b, Apparent turnover rates k_cat(app) for the conversion of 8 to the adducts by the mutants for examining the role of active-site residues. The centre value is the mean of triplicate measurements (open circles) with the error bar representing the standard deviation. c, relative production levels of 9 versus 10 by different mutants for exploring the roles of octalin-packing residues. See Methods and Supplementary Note 5 for details.

Fig. 4 |. Computational analyses of the transition states and energetics of the octalin-forming intramolecular Diels–Alder reactions.

a, Steps in the CghA-catalysed reaction leading to endo 1 and exo 4 adducts from enol-form (Z)-tetramate R-1 via enol-form (E)-tetramate R-1′ formed upon rotation of the tetramate moiety. TS-1 and TS-2 are the transition states for the endo and exo pathways, respectively. The non-enzymatic reactions occur directly from R-1 via TS-1 and TS-2. b, The computed transition state structures and the energy diagram of the reaction pathways. Energies were calculated in the gas phase (black) and using the CPCM implicit water model (blue).