SyrB2 in syringomycin E biosynthesis is a nonheme FeII alpha-ketoglutarate- and O2-dependent halogenase

Abstract

The nine-residue lipodepsipeptide syringomycin E, elaborated as a phytotoxin by Pseudomonas syringae pv. syringae B301D contains a 4-Cl-L-Thr-9 moiety where failure to chlorinate results in a 3-fold drop in biological activity. The proteins SyrB1 and SyrB2 encoded by the biosynthetic cluster are shown to act as a substrate and enzyme pair for SyrB2-mediated chlorination of the aminoacyl-S-enzyme L-Thr-S-SyrB1. SyrB2 is a member of the nonheme Fe(II) alpha-ketoglutarate-dependent enzyme superfamily, and requires O2 and alpha-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH3 group of L-Thr-S-SyrB1. Chlorination of L-Thr-S-SyrB1 was validated by thioesterase-mediated release of L-Thr and 4-Cl-L-Thr, N-derivatization as fluorescent isoindoles, and HPLC separation compared with authentic standards. Incubations with L-[14C]Thr and [36Cl-] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme Fe(II) enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.

Fig. 1.

Structures of chlorine-containing natural products. (A) Aromatic and heteroaromatic natural organohalogens. (B) Aliphatic natural organohalogens containing one, two, or three chlorines at a single carbon center.

Fig. 2.

l-Thr activation and self-loading reaction catalyzed by SyrB1. (A) Reaction catalyzed by SyrB1. A, adenylation domain of SyrB1; T, thiolation domain of SyrB1. (B) Self-loading reaction catalyzed by SyrB1. Lane 1 and 4, molecular mass standards. Lane 2, holo-S-SyrB1, l-[¹⁴C]Thr, ATP. Lane 3, holo-S-SyrB1, l-[¹⁴C]Thr, no ATP.

Fig. 3.

Formation of 4-Cl-l-Thr by SyrB2. (A) HPLC traces of hydrolyzed amino acid obtained after incubation of l-[¹⁴C]Thr-S-SyrB1 ± SyrB2. (B) Mass spectrometric analysis of the isoindole derivatives of l-Thr and 4-Cl-l-Thr hydrolyzed from SyrB1.

Fig. 4.

Dependencies of the SyrB2 reaction. (A) HPLC traces of hydrolyzed amino acid obtained after incubation of l-Thr-S-SyrB1 with SyrB2. (Aa) Reaction performed with l-Thr-S-SyrB1, α-KG and SyrB2 in presence of Na[³⁶Cl]. (Ab) Reaction performed with l-[¹⁴C]Thr-S-SyrB1, chloride, α-KG, and SyrB2. (Ac) Reaction performed with l-[¹⁴C]Thr-S-SyrB1, chloride, and SyrB2 in the absence of α-KG. (Ad) Reaction performed with l-[¹⁴C]Thr-S-SyrB1, α-KG, chloride, and apo-SyrB2 (< 5% Fe^II). (Ae) UV trace of l-Thr and 4-Cl l-Thr standards. (B) Fluorescence HPLC traces showing the oxygen dependency of the SyrB2 reaction. (Ba) Reaction performed with l-Thr-S-SyrB1, chloride, α-KG, and SyrB2 in air. (Bb) Reaction performed with l-Thr-S-SyrB1, chloride, α-KG, and SyrB2 in the absence of oxygen. (Bc) UV trace of l-Thr and 4-Cl l-Thr standards. (C) Incorporation of [³⁶Cl]chloride into l-Thr-S-SyrB1 by SyrB2. Lane 1 and 5, molecular mass standards. Lane 2, l-Thr-S-SyrB1, Na[³⁶Cl], and SyrB2. Lane 3, holo-S-SyrB1, Na[³⁶Cl], and SyrB2. Lane 4, l-Thr-S-SyrB1 and Na[³⁶Cl]. α-KG and O₂ were present in all assays (lanes 2-4).

Fig. 5.

Proposed mechanism of halogenation catalyzed by SyrB2 involving formation of the Fe^IV═O intermediate characteristic of this enzyme class but its diversion for chlorination rather than hydroxylation. R-CH₃ is the C₄ methyl group of the l-Thr-S-pantetheinyl-SyrB1 protein substrate.