Dichlorination and bromination of a threonyl-S-carrier protein by the non-heme Fe(II) halogenase SyrB2.

Biosynthetic tailoring of nonribosomal peptide and polyketide natural products can enhance their biological activities. Tailoring enzymes can introduce alkyl, acyl, or glycosyl groups onto natural product scaffolds and can oxidize or halogenate biosynthetic intermediates. Chlorinated and brominated molecules make up more than 95% of the more than 4500 known halogenated metabolites. Chloro and bromo substituents are frequently found on aromatic and heteroaromatic rings, and many terpene scaffolds are also brominated and chlorinated by marine microorganisms. Halogenating enzymes discovered to date fall into two categories based on their utilization either of hydrogen peroxide (haloperoxidases) or molecular oxygen (halogenases) as required oxidants. Haloperoxidases can contain either heme iron or a vanadate cofactor, thought to generate enzyme-bound hapohalite equivalents as proximal OCl or OBr. 8] The O2-utilizing halogenases are typically found embedded in biosynthetic gene clusters ; this suggests a tailoring role in specific natural product assembly. This second class of enzymes uses either FADH2 or non-heme Fe II to activate chloride or bromide oxidatively. The flavoproteins work on electron-rich aromatic and heteroaromatic substrates. 12] The Fe halogenases represent a new branch of the O2 and a-ketoglutarate-decarboxylating superfamily and are powerful enough to halogenate unactivated carbon centers on aminoacyl groups tethered to nonribosomal peptide synthetase assembly lines. Thus, the 4-Cl-l-Thr residue in the phytotoxic liACHTUNGTRENNUNGpo ACHTUNGTRENNUNGdepsipeptide syringomycin E (1) is generated by the nonheme Fe halogenase SyrB2 (Scheme 1). Chlorination occurs on the threonyl skeleton only while it is linked via a thioester to a peptidyl carrier protein domain. Remarkably, the cyclopropane ring in the amino acid coronamic acid arises by a similar g-chlorination of an l-allo-Ile-S-protein by the halogenase CmaB. The g-chloride is then displaced intramolecularly by a thioester enolate by action of CmaC. Therefore, the CmaBmediated chlorination is cryptic in cyclopropane formation. In surveying natural products in which biological chlorination is likely to have occurred at an unactivated carbon center, the remarkable functionalization of the two prochiral methyl groups of leucine to yield the regioand stereospecific generation of a trichloromethyl group in the biosynthesis of the cyanobacterial metabolites barbamide (2), dysidenin (3), and dysideathiazole (4 ; Scheme 2A) suggests analogies with the above Fe halogenases. Indeed, the barbamide biosynthetic gene cluster has been sequenced and contains two genes (barB1 and barB2) that encode proteins homologous to SyrB2, but no activity has yet been reported. barB1 and barB2 homologues have also been found in the dysidenin and dysideathiazole producers (dysB1/dysB2 ; one pair in each producer). Only a few natural products contain bromine where biological bromination might have occurred at an unactivated carbon site. One example is lyngbyaloside B (5 ; Scheme 2B), which is also of cyanobacterial origin and could arise from bromination of an unactivated carbon on a biosynthetic precursor. Whether nonheme Fe halogenases are involved in the biosynthesis of brominated natural products is unknown. To evaluate whether the non-heme Fe family of enzymes can indeed carry out bromination and iterative chlorinations at the same carbon site, we have further examined the activity of SyrB2. SyrB2 was shown to act on the threonyl group presented in thioester linkage on the peptidyl carrier protein domain of its partner protein SyrB1. 4-Cl-threonyl-S-SyrB1 was gently hydrolyzed by addition of the thioesterase TycF and detected as the isoindole adduct (Figure 1A). With an almost equimolar ratio of SyrB2/SyrB1, SyrB2 generated a new peak (Figure 1B) that coeluted with the isoindole derivative of authentic 4,4diCl-Thr, synthesized as noted in the Experimental Section. Mass analysis of the new enzymatic product confirmed both the mass and isotope ratios of the diCl-l-Thr isoindole derivative (calcd for [M+H] 392.0 (100%), 394.0 (71%); found 391.7 (100%), 394.1 (68%)). In addition, the ratio of the relative intensity of the peaks corresponding to 4-Cl-l-Thr and 4,4-diCl-lThr for the reaction run in the presence of [Cl] is doubled in the radioactivity detection channel (Figure 1B, trace c) when compared to the UV channel (Figure 1B, trace d) of the same reaction; this is in good agreement with the presence of two chloro substituents. Because the substrate for SyrB2 is a covalent aminoacyl-S protein and the chlorinated product(s) remain covalently tethered, kinetic analysis would be challenging. However, the released monoand diCl-l-Thr products were obtained at a maximum ratio of about 0.38:1 diCl-l-Thr/Cl-l-Thr (Figure 1C). When comparing the ratio of diCl-l-Thr/Cl-l-Thr in Figure 1C and B, it can be seen that the ratio is higher for panel B (1:1 diCl-l-Thr/Cl-l-Thr). This seems to be due to the difference in initial oxygen concentration prior to SyrB2 addition. In panel B, the reaction mixture was anaerobic prior to SyrB2 addition, whereas the buffer was air-saturated for panel C. The slow addition of oxygen after SyrB2 addition increases the product yield, as shown in Figure 1B. This was also observed when demonstrating the oxygen dependency of the reaction in a prior study (Figure 2B in ref. [10]), in which full [a] Dr. F. H. Vaillancourt, Prof. Dr. D. A. Vosburg, Prof. C. T. Walsh Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 (USA) Fax: (+1)617-432-0438 E-mail : [email protected] [b] Dr. F. H. Vaillancourt Present address: Department of Biological Sciences Research and Development, Boehringer Ingelheim (Canada) Ltd Laval, QC, H7S 2G5 (Canada) [c] Prof. Dr. D. A. Vosburg Present address: Department of Chemistry, Harvey Mudd College Claremont, CA 91711 (USA) [] These authors contributed equally to this work.