Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis

Abstract

The medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]-spiroketal pharmacophore that was recently shown to be assembled by flavin-dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]-spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon-carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals.

Keywords: antibiotics; collinone; lenticulone; polyketide synthase; redox tailoring.

Figure 1

Detailed reaction mechanism proposed for GrhO5 (orange box), GrhO1 and GrhO6 leading to the [5,6] spiroketal containing rubromycin polyketides. After the initial reduction of 3 to 5, GrhO5 is expected to catalyze an aromatic hydroxylation reaction, leading to the formation of unstable secocollinone (10, that required methylation prior to structural characterization due to high innate reactivity ^[6] ). Compound 10 is further converted into 6, which may either autooxidize and decarboxylate to 4 (that once more autooxidizes to lenticulone (9), analogous to the spontaneous formation of 3 from 5), or decarboxylate and autooxidize to shunt product 7. Formation of the shunt product is suppressed by GrhO1, which likely oxidizes ring B of compound 6 to boost 4 formation. Finally, GrhO6 converts 4 into the [5,6]‐spiroketal containing compound 7,8‐dideoxy‐6‐oxo‐griseorhodin C (8) that is further processed by pathway‐specific enzymes (not shown) to the mature rubromycins (red box). The numbered compounds were previously identified and (partially) structurally characterized (other intermediates are postulated). For details on individual steps, see also main text.

Figure 2

Overall structure of GrhO5 (A) and close‐up view of the NADPH‐ (B, C) and the substrate‐entry sites (D, E) in GrhO5 and RubL, respectively. A, Domain I (gold; residues 1–77, 108–188, and 287–386), domain II (blue; residues 78–107 and 189–286), and domain III (pink; residues 387–537). B/C, The NADPH‐entry site. This entry site is blocked by the W281 side chain (W289 for RubL) in ligand‐free (acceptor state; B) and 3/5‐bound GrhO5 (reducible state). Upon rotation of this particular tryptophan residue, as observed in the crystal structure of RubL (catalytic state, C), the NADPH‐entry site opens up, thereby probably promoting the expulsion of NADP⁺. D/E, Substrate‐entry site. The substrate‐entry site is located on the opposite side of the enzymes and is open both in ligand‐free (acceptor state) and 3/5‐bound GrhO5 (reducible state; D) due to an unordered loop comprising residues 90–110. However, when the protein adopts the catalytic state (E), this mobile loop becomes ordered (highlighted in lilac) and closes the substrate entry‐site.

Figure 3

Comparison of group A FPMOs including GrhO5‐like type I (orange), MtmOIV‐like type II (red), and PHHY‐like (blue). A, Close‐up views of the active sites with FAD in “OUT” (GrhO5, PHHY) or “IN” (MtmOIV; “OUT” structure not available). B, Structure‐based sequence alignment of selected group A FPMOs. C, Visualization of the phylogenetic relationships using a bootstrapped distance tree (1000 iterations). See Table S2 for pdb codes and related information and ref.  for a comprehensive tree of the entire group A FPMO family.

Figure 4

Close‐up view of the substrate binding sites (A, B), the O₂ reaction site (C) and the FAD surroundings (D) of GrhO5/RubL. The active‐sites of GrhO5 (A, C) and RubL (B, C) are shown in the “reducible state” (3‐soaked GrhO5 with FAD in OUT (yellow); A, C) and the “catalytic state” (RubL with the FAD in IN (orange) and 3 modeled into the active site; B, C), respectively. Panel C shows an overlay of A and B to illustrate different positions of active site residues and the proposed O₂ reaction site (Cl⁻ binding site, red sphere). Compound 3/5 (purple) and selected active‐site residues (grey and teal for GrhO5 and RubL, respectively) are shown as sticks. Electrostatic interactions (<3.8 Å distance) between several amino acid side chains, the FAD cofactor, and 3/5 are indicated by dashed lines. D, FAD surroundings in the acceptor state (grey), the reducible state (purple) and the catalytic state (teal). The movement of FAD to IN is accompanied by changes in the rotameric state of F279 (RubL F287) and H43 (RubL H51) that move toward the center of the active site, resulting in a change of their T‐shaped π‐π‐ to a parallel displaced π‐π stacking interaction in the catalytic state.

Figure 5

Spectral properties of RubL. A, UV‐visible absorption spectra of native (black line) and denatured (red line) RubL. B, Selected absorption spectra recorded in the course of the anaerobic photoreduction of the RubL‐bound FAD cofactor (top black line, start spectrum; top yellow line, spectrum recorded after reoxidation). The intensities of the peaks at 370 and 450 nm decrease simultaneously, suggesting a direct two‐electron reduction of FAD that goes along with the loss of the CT interaction (shown enlarged in the Inset).

Figure 6

Proposed mechanistic scheme for the conversion of 3 (dark purple) into final product 6 via 5 (yellow) and 10 (light yellow) involving a mobile FAD that adopts the OUT or IN position. The FAD is colored according to its redox state (FAD_ox, orange; FAD_red, beige; FAD_C4aOOH, red). A proposed FAD‐catalyzed two‐electron oxidation during formation of 6 from 10 is not shown (see Figure 1). A, Cartoon representation of proposed FAD movements and redox catalysis, see text for details on each step. B, Redox states adopted by FAD in the course of the hydroxylation of the phenolate moiety of 5.