Structure and stereospecificity of the dehydratase domain from the terminal module of the rifamycin polyketide synthase

Abstract

RifDH10, the dehydratase domain from the terminal module of the rifamycin polyketide synthase, catalyzes the stereospecific syn dehydration of the model substrate (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, resulting in the exclusive formation of (E)-2-methyl-2-pentenoyl-RifACP10. RifDH10 does not dehydrate any of the other three diastereomeric, RifACP10-bound, diketide thioester substrates. On the other hand, when EryACP6, from the sixth module of the erythromycin polyketide synthase, is substituted for RifACP10, RifDH10 stereospecifically dehydrates only (2R,3R)-2-methyl-3-hydroxypentanoyl-EryACP6 to give exclusively (E)-2-methyl-2-pentenoyl-EryACP6, with no detectable dehydration of any of the other three diastereomeric, EryACP6-bound, diketides. An identical alteration in substrate diastereospecificity was observed for the corresponding N-acetylcysteamine or pantetheine thioester analogues, regardless of acyl chain length or substitution pattern. Incubation of (2RS)-2-methyl-3-ketopentanoyl-RifACP10 with the didomain reductase-dehydratase RifKR10-RifDH10 yielded (E)-2-methyl-2-pentenoyl-RifACP10, the expected product of syn dehydration of (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, while incubation with the corresponding EryACP6-bound substrate, (2RS)-2-methyl-3-ketopentanoyl-EryACP6, gave only the reduction product (2S,3S)-2-methyl-3-hydroxypentanoyl-EryACP6 with no detectable dehydration. These results establish the intrinsic syn dehydration stereochemistry and substrate diastereoselectivity of RifDH10 and highlight the critical role of the natural RifACP10 domain in chaperoning the proper recognition and processing of the natural ACP-bound undecaketide substrate. The 1.82 Å resolution structure of RifDH10 reveals the atomic-resolution details of the active site and allows modeling of the syn dehydration of the (2S,3S)-2-methyl-3-hydroxyacyl-RifACP10 substrate. These results suggest that generation of the characteristic cis double bond of the rifamycins occurs after formation of the full-length RifACP10-bound acyclic trans-unsaturated undecaketide intermediate, most likely during the subsequent macrolactamization catalyzed by the amide synthase RifF.

Figure 1

RifDH10 dehydrates a RifACP10-bound (2S,3S)-2-methyl-3-hydroxyacyl undecaketide generated by the paired ketoreductase domain RifKR10 in the terminal module 10 of the rifamycin PKS. The amide synthase RifF catalyzes the macrolactamization of the acyclic undecaketide product to the rifamycin B precursor, proansamycin X, either before or after isomerization to form the characteristic cis double bond.

Figure 2

RifDH10-catalyzed dehydration/hydration of RifACP10-bound substrates a) Dehydration of (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10 (1a) b) Hydration of (E)-2-methyl-2-pentenoyl-RifACP10 (2). c) Incubation of Rif[DH10+KR10] with RifACP10- and EryACP6-bound 2-methyl-3-ketoacyl thioesters.

Figure 3

Chiral GC-MS analysis (Method 1) of the incubation of (2RS)-2-methyl-3-ketopentanoyl-RifACP10-NusA with RifKR7 and RifDH10. A and D: (E)-2-Methyl-2-pentenoic acid (3) from RifKR7-catalyzed reduction of (2RS)-2-methyl-3-ketopentanoyl-RifACP10-NusA followed by dehydration by RifDH10. B: (Z)-2-methylpentenoic acid authentic standard. C: Co-injection of A with B. E: Co-injection of (E)-2-methyl-2-pentenoic acid standard with D. A-1, B-1, C-1, D-1 and E-1: Extracted ion current (XIC) at m/z 114 (base peak). A-2, B-2, C-2, D-2 and E-2: mass spectra of selected peak, upper half, observed spectrum, lower half, inverted mass spectrum of reference standard.

Figure 4

Chiral GC-MS analysis of the incubation of (2RS)-2-methyl-3-ketopentanoyl-RifACP10-NusA with RifDH10-KR10 (Methods 2 and 4). Left panels: A1. Extracted ion current (XIC) at m/z 88 (base peak for 4a) (Method 4); B-1 and C-1. Extracted ion current (XIC) at m/z 114 (base peak for 3) (Method 2). Right panels, mass spectra of selected peaks, upper half, observed spectrum, lower half, inverted mass spectrum of reference standard. A. (2S,3S)-4a produced by RifDH10-KR10. B. (E)-2-Methylpentenoic acid (3) produced by RifDH10-KR10.

Figure 5

Chiral GC-MS analysis (Method 4) of the incubation of (E)-2-methyl-2-pentenoyl-RifACP10-NusA with RifDH10. Left panels: Extracted ion current (XIC) at m/z 88 (base peak). Right panels, mass spectra of selected peaks corresponding to diastereomers of methyl 2-methyl-3-hydroxypentanoate, upper half, observed spectrum, lower half, inverted mass spectrum of reference standard. A and C. (2S,3S)-4a generated by RifDH10. B. A plus (2R,3R)-4b. D. C plus (2S,3S)-4a. Detailed analysis of the full mass spectra of all peaks detected in Panel A1 between 31.00 and 34.00 min showed only the presence of the single (2S,3S)-4a.

Figure 6

RifDH10-catalyzed dehydration/hydration of acyl thioester analogues. a) Dehydration of (2R,3R)-2-methyl-3-hydroxypentanoyl-EryACP6 (5b). b) Hydration of (E)-2-methyl-2-pentenoyl-EryACP6 (6). c) Dehydration of (2R,3R)-2-methyl-3-hydroxypentanoyl-S-Pant (7b) and –S-NAC (9b) analogues. d) Incubation of RifDH10 with (E)-11 and (Z)-13.

Figure 7

The 1.82 Å-resolution structure of RifDH10. a) The 2F_o-F_c electron density map (contoured at 1.8 σ) shows a water molecule, bound to the catalytic histidine (H50) and aspartate (D220), as well as a nearly invariant Y174, that is representative of the water molecule eliminated through a syn-dehydration reaction. H224 and Y164 may help increase the pK_a of D220 through a hydrogen-bond network. b) Stereodiagram of the superposition of the active sites of RifDH10, EryDH4, CurF DH, CurH DH, CurJ DH, and CurK DH, showing the catalytic His (H) and Asp (D) residues.

Figure 8

RifDH10-catalyzed dehydration. a) syn-Dehydration of a (2S,3S)-2-methyl-3-hydroxyacyl thioester substrate generates a trans (E) double bond. b) Stereodiagram showing (2S,3S)-2-methyl-3-hydroxyacyl thioester substrate modeled into the active site of RifDH10. See Figure S2 for modeling of diastereomeric thioesters into the active site.