Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis

Abstract

The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH2. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCl intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.

Fig. 1.

Flavin-dependent halogenases in natural product biosynthesis. (A) Natural products containing chlorinated aromatic and heteroaromatic rings. (B) Formation of the dichloropyrrole moiety during pyoluteorin biosynthesis

Fig. 2.

Chlorination of pyrrolyl-S-PltL by PltA. (A) Comparison of the theoretical isotopic distribution to the experimental data for prolyl-S-PltL. (B) Comparison of the theoretical isotopic distribution to the experimental data for dichloropyrrolyl-S-PltL. (C) Acyl-PltL species resulting from a reaction containing pyrrolyl-S-PltL, FAD, chloride, SsuE, PltA, PltM, and NADH were separated by HPLC and analyzed by ESI-FTMS. MS analysis of the fraction eluting between 19.2 and 19.4 min is shown and contains a product whose mass corresponds to the dichloropyrrolyl-S-PltL (11,634.2 Da). (D) The same reaction as in C but with NADH omitted. (E) The same reaction as in C but with SsuE omitted. (F) The same reaction as in C but with PltA omitted. (G) The same reaction as in C but with both PltA and PltM omitted. (H) The same reaction as in C but with PltM omitted. The charge of the ions in A and B is 13+; the charge state for all other ions shown is 10+.

Fig. 3.

Localization of the dichlorination on PltL by tandem MS. (A) The protein sequence of PltL is shown in which Ser-42 (shaded) is covalently modified by a phosphopantetheinyl arm acylated with dichloropyrrole. b-fragments (gray brackets) and y-fragments (black brackets) resulting from collisionally activated dissociation of the dichloropyrrolyl-S-PltL species are indicated. Numbers designate specific y-fragments whose mass spectra are shown below. (B) Mass spectra of fragments y56, y57, and y58 that contain Ser-42 (and the dichloropyrrolyl modification) have the characteristic isotopic pattern where the +2-Da isotope is as large or larger than the parent isotope because of the incorporation of two chlorines. Fragments y54, y53, and y47 lacking this residue do not display this characteristic pattern. For all fragments shown, the +2-Da isotope is indicated (*).

Fig. 4.

Regiospecific chlorination at C5 on the pyrrole ring followed by chlorination at C4. [¹⁴C]pyrrolyl-S-PltL substrate (derived from l-[¹⁴C]proline) was prepared. After reaction with PltA and SsuE from 0 to 40 min, reaction products were released from the protein by thioesterase treatment and analyzed by radio-HPLC. The observed products were compared with chemical standards of pyrrole-2-carboxylic acid, 5-chloropyrrole-2-carboxylic acid, 4-chloropyrrole-2-carboxylic acid, and 4,5-dichloropyrrole-2-carboxylic acid, which were also separated by HPLC (bottom trace; monitored at 260 nm).

Fig. 5.

Mechanistic proposal for the formation of dichloropyrrolyl-S-PltL. A proposed FAD-4a-OCl intermediate is formed from the attack of Cl^– on FAD-4a-OOH. The FAD-4a-OCl intermediate then reacts with substrate pyrrolyl-S-PltL in a two-electron electrophilic aromatic substitution to first form the 5-chloropyrrolyl intermediate and then the 4,5-dichloropyrrolyl product.