Cartoon representation of the proposed proton-relay mechanism. The three docking points for intermediate 1 are labeled in the order of the proton-relay event. The active site consists of NADPH and the catalytic tetrad N114-S144-Y157-K161. Hydrogen bonds are in cyan.

Enzyme kinetics of actKR. (A) The double reciprocal plot (1/v versus 1/[trans-1-decalone]) altering the concentration of trans-1-decalone and NADPH eliminates a ping-pong mechanism (which will produce parallel lines) as a possible mechanism. (B) The double reciprocal plot that changes the concentration of the inhibitor AMP, NADPH, and trans-1-decalone indicates that AMP is a competitive inhibitor (right) of NADPH but a noncompetitive inhibitor for trans-1-decalone (left). (C) The double reciprocal plot that changes the concentration of the inhibitor emodin, NADPH, and trans-1-decalone indicates that emodin is a competitive inhibitor of trans-1-decalone (left) but a uncompetitive inhibitor for NADPH (right). The results from A, B, and C indicate that actKR proceeds via an ordered Bi Bi mechanism.

(A and B) The wild type actKR-NADPH-emodin cocrystal structure shows that the quinone carbonyl group of emodin is in the three-point docking position. Surprisingly, the p-quinone is bent, as seen in the 3F_o –2F_c density contoured at 1σ. In addition, an unbiased 2F_o − F_c simulated annealing omit map (not shown) in this region contoured at 1σ also shows partial density for the core of the bent emodin, confiming the bent geometry. (B) With the view rotated 90°. (C and D) Similarly, the wild type actKR-NADP⁺-emodin cocrystal structure shows that the quinone carbonyl group of emodin is in the three-point docking position (2F_o − F_c density map is shown, contoured at 0.6s), and the p-quinone is also bent, as shown in D with the view rotated 90°. (E and F) In comparison, the P94L actKR-NADPH-emodin cocrystal structure shows a flat p-quinone in the 2F_o − F_c simulated annealing omit map contoured at 1s. Also, unexpectedly, there are two molecules of emodin bound in the P94L mutant active site with the first molecule (purple) at full occupancy while there is only partial density in the sa-omit map for the second molecule when also modeled at full occupancy (density map not shown). This result indicates that a change in the actKR active site geometry affects substrate/inhibitor binding, and also that the observed bent emodin in the wild type is not a crystallization defect. The electron density surrounding emodin is shown in blue mesh.

(A) The actinorhodin biosynthesis pathway. The polyketide chain is first assembled by the minimal PKS (acyl-carrier protein ACP, ketosynthase KS, and chain length factor CLF), followed by reduction by KR at the C9 position and cyclization/aromatization of the polyketide chain. (B) The in vitro actKR substrate candidates 7–16 that were tested in this work. actKR accepts only the bicyclic substrates 7–9 in vitro. (C) Intermediate 5, its aromatized form, and the tautomers. (D) The inhibitor emodin 17 and its tautomers.

Docking simulation between actKR and trans-1- or 2-decalone. (A) trans-1-Decalone binds tightly to actKR with a single predicted conformation. (B) 2-decalone binds less tightly to actKR with multiple possible binding modes.

(A) The tetramer (in different colors) cocrystal structure of wild type actKR-NADPH-emodin, in which the cofactor NADPH (in sticks) is bound to all four monomers, whereas well-defined electron density of emodin (in spheres) can be found in monomers A and C. (B) The asymmetric unit contains monomers A (middle panel) and B (left panel) in open and close conformations, respectively. Surface potentials were colored from negative (red) to positive (blue). When the open and closed conformations are overlapped (right panel, open in yellow, close in purple), the major conformational change is in the flexible α6-α7 region, with a 7.9 Å difference between the two monomers.

Multiple docking rounds of possible actKR intermediates (1 and 5 in Figure 1A) show high consistency in docking results. (A) In the hydride transfer site, the C9 carbonyl groups of the monocyclic 1 (magenta) and the bicyclic 5 (yellow) are consistently docked to the optimal position for ketoreduction. Further, at least the constraints from the first ring are necessary to properly orient the substrate for C9 reduction. (B) The phosphopantetheine (PPT) group, which is tethered between acyl carrier protein and the polyketide substrate, is consistently docked into a groove on the actKR surface, where the positively charged groove interacts with the PPT phosphate group.