Characterization of the Initial Enzymatic Steps of Barbamide Biosynthesis

Abstract

Barbamide is a mixed polypeptide-polyketide natural product that contains an unusual trichloromethyl group. The origin of the trichloromethyl group was previously shown to be through chlorination of the pro-R methyl group of L-leucine. Trichloroleucine is subsequently decarboxylated and oxidized to trichloroisovaleric acid and then extended with an acetate unit to form the initial seven carbons of barbamide. In this study we used a combination of biosynthetic feeding experiments and enzymatic analysis to characterize the initial steps required for formation of trichloroleucine and its chain-shortened product, trichloroisovaleric acid. Results from isotope-labeled feeding experiments showed that both dichloroleucine and trichloroleucine are readily incorporated into barbamide; however, monochloroleucine is not. This suggests that halogenation of the pro-R methyl group of leucine occurs as two discrete reactions, with the first involving incorporation of at least two halogen atoms and the second converting dichloroleucine to trichloroleucine. Additionally, the initial tandem dichlorination must occur before substrate can be further processed by the remainingbar pathway enzymes. In vitro analysis of the first five open reading frames (ORFs; barA, barB1, barB2, bar C, barD) of the barbamide gene cluster has yielded new insights into the processing of leucine to form the trichloroisovaleryl-derived unit in the final product.

Biosynthetic origin of natural products isolated from marine microorganism–invertebrate assemblages

“…The halogen atoms comprising the unique trichloromethyl group are located at a position without an adjacent activating functionality, and this suggested a novel route for their biochemical incorporation (85,86). These barbamide halogenases and related enzymes have since been characterized as a new class of ''radical halogenases'' capable of direct halogenation of unreactive carbon atoms in natural products, such as methyl groups (87)(88)(89). Early on, we recognized the striking structural similarity of barbamide to a number of Dysidea''sponge'' chlorinated peptides.…”

Developing Streptomyces venezuelae as a cell factory for the production of small molecules used in drug discovery

“…Barbamide is a chlorinated lipopeptide that has been isolated from a Curaçao strain of cyanobacterium Moorea producens. The structure of barbamide has several unique biochemical features, such as trichlorination, a one-carbon truncation during chain extension, an E-double bond, and a thiazole ring (Orjala and Gerwick 1996;Flatt et al 2006). It was isolated as an active toxin component from the extract of cyanobacteria which shows toxicity against mollusc Biomphalaria glabrata (LC 100 of 10 lg/ml).…”

Synthesis of Halogenated α‐Amino Acids

“…It has been shown that selective chlorination of the pro-(R) methyl group of leucine occurs leading via dichloroleucine 72 to trichloroleucine 77 prior to incorporation into the marine natural product (Scheme 10.18). In 2006, a novel iron-dependent mechanism of biochlorination at unactivated positions was described by the groups of Walsh [48] and Gerwick [49], giving an insight into these fascinating transformations. 3-Trichloromethylbutanoic acid 78 has been used in the synthesis of such compounds and may be prepared in a number of ways.…”