Structure and Function of a Dual Reductase-Dehydratase Enzyme System Involved in p-Terphenyl Biosynthesis

Abstract

We report the identification of the ter gene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomyces strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis.

Figure 1.

Examples of microbial p-terphenyls (A) and bis(indolyl)benzoquinone compounds (B). Structures in blue are metabolites isolated from Appalachian Streptomyces RM-5–8.

Figure 2.

Biosynthesis of terfestatins. (A) Putative ter BGC from Streptomyces RM-5–8 highlighting terB and terC (red) and (B) proposed biosynthetic pathway for terfestatins.

Figure 3.

Conversion of PPA (2) to 3 under different conditions. (A) HPLC chromatograms of reactions performed in Tris 50 mM pH 8.0 containing 0.4 mM 2 and different combinations of 10 mM NADH and/or 6.6 μM of each of TerB and/or TerC as following: (i) NADH, (ii) NADH/TerB, (iii) NADH/TerC, (iv) TerB/TerC, (v) NADH/TerB/TerC, and (vi) NADH/TerB/TerC spiked with 3; conversion percentage of 2 to 3 in the presence of increasing concentrations of (B) 0.4, 2, and 20 mM NADH and NADPH in the presence of wild-type (wt) TerB and wt TerC; and (C) 2 and 20 mM NADH and NADPH in the presence of wt TerB and wt TerC, TerC_R143A, TerC_H159A, or TerC_D173A. The reactions were performed in Tris 50 mM pH 8.0 containing 0.4 mM PPA, 6.6 μM wt TerB, and 6.6 μM wt or mutant TerC. All samples were incubated at 30 °C for 60 min.

Figure 4.

X-ray crystal structures of substrate-bound TerB and TerC. (A) Overall structure of TerB (cyan) with 2 and NADPH shown as sticks in their binding sites. Residues that interact with the ligands shown as sticks. (B) 2fo–fc Density for the 2 and NADPH ligands shown at 2RMSD. Both ligands are very well resolved and the phosphate group differentiating NADPH from NADH is clearly present. (C) Active site and surrounding residues of interest. Compound 2 is recruited by W141, Y181, and Y188 with R268 latching above 2. (D) TerC jelly-roll fold and residue sidechains exposed to the binding site at the center of the protein (magenta) are shown around the substrate species of 2 (gray) and the interesting water molecule (red sphere). (E) TerC active site showing distances between the conserved Asp and Arg residues, water molecule, His nitrogen atom, and 2. (F) 2fo-fc Density of the active site at 1RMSD.

Figure 5.

Proposed mechanism for roles of TerB (A) and TerC (B) in the conversion of 2 to 3. B denotes Arg143 or Asp173 side chain participation. Based on the determined TerC structure, some steps may be H₂O-mediated.