Characterization of a novel type of oxidative decarboxylase involved in the biosynthesis of the styryl moiety of chondrochloren from an acylated tyrosine

Abstract

Myxobacteria are soil-dwelling bacteria notable for several unique behavioral features, such as cellular movement by gliding and the formation of multicellular fruiting bodies. More recently they have gained recognition as producers of several unique polyketide and nonribosomal polypeptide metabolites with potential therapeutic value. The biosynthesis of these compounds often involves highly unusual mechanisms including the formation of the chloro-hydroxy-styryl moiety of the chondrochloren antibiotic produced by Chondromyces crocatus Cm c5. Here it is shown that the final product of the chondrochloren megasynthetase is the novel natural product pre-chondrochloren, a carboxylated and saturated derivative of chondrochloren. This compound was isolated from strains harboring mutants of a hypothetical oxidative decarboxylase (CndG) identified in the chondrochloren gene cluster. CndG was heterologously expressed in Escherichia coli and shown to be an FAD-dependent oxidative decarboxylase. Biochemical characterization of the protein was achieved using the intermediate described above as the substrate and yielded chondrochloren by oxidative decarboxylation. It was also demonstrated that the CndG post-assembly line modification of pre-chondrochloren is essential for the biological activity of chondrochloren.

FIGURE 1.

Oxidative decarboxylation steps proposed for the biosynthesis of barbamide catalyzed by BarJ (a), fatty acid enol ester of the fee gene cluster by FeeG (b), and chondrochloren (c). CndG is shown here to decarboxylate the pre-chondrochlorens A ((3)) and pre-chondrochloren B ((4)) to yield the mature chondrochloren A ((1)) and chondrochloren B ((2)), respectively.

FIGURE 2.

Inactivation of the cndG gene in Cm c5 by insertion of the pSR13 plasmid into the bacterial genome via single cross-over, creating Cmc-cndG⁻. a, schematic representation of the pSR13 plasmid, the genomic chondrochloren region in the wild type and in the Cmc-cndG⁻ mutant. b, verification of the resulting mutants, showing amplification of a 1016-bp PCR fragment (using the oligonucleotides PSUP-EV and FAD-XhoI-dn) in three isogenic mutants (lanes 3–5), in comparison with the wild type (lane 2). Lane 7 shows amplification of a 1639-bp PCR fragment of the intact cndG (using the oligonucleotides FAD-EcoRI-up and FAD-XhoI-dn) in the wild type in comparison with the mutants (lanes 8–10). Lanes 1 and 6, 1-kb DNA marker (Fermentas). c, HPLC chromatogram of chondrochlorens A (peak 1) and B (peak 2) production by Cm c5. d, HPLC analysis of the Cmc-cndG⁻ mutant, showing disappearance of chondrochloren peaks.

FIGURE 3.

a and b, HPLC-MS analysis of cell-free extracts of the Cm c5 wild type (a) and the Cmc-cndG⁻ mutant (b), showing peaks 3 and 5 (pre-chondrochloren A isomers) and peaks 4 and 6 (pre-chondrochloren B isomers) in Cm c5 and in the mutant. c, mass spectrum of the pre-chondrochloren A (peak 3) (R_t = 17.8 min, m/z [M + Na]⁺ = 594.3). d, mass spectrum of carboxylated chondrochloren B (peak 4) (R_t = 22.0 min, m/z [M + Na]⁺ = 608.3).

FIGURE 4.

NMR analysis and structure elucidation of the carboxylated chondrochloren B and heteronuclear multiple bond correlations found for compound 4.

FIGURE 5.

Purification of recombinant CndG. a, lane 1, protein marker. Lane 2, whole cell extract of induced E. coli BL21, containing pGEX-CndG. Lane 3, purification of GST-tagged CndG (asterisk) by glutathione affinity chromatography. Lane 4, purified CndG (asterisk), following treatment of the GST fusion protein with PreScission Protease. b, conversion of pre-chondrochlorens 3 and 4 to the mature chondrochlorens 1 and 2, respectively, catalyzed by in vitro action of CndG, monitored by HLPC analysis over a 30-min incubation period.

FIGURE 6.

a and b, oxidative decarboxylation by CndG under aerobic (a) and anaerobic (b) conditions. Under anaerobic conditions, production of compound 2 from its carboxylated counterpart was essentially undetectable, whereas the yield of compound 1 was reduced by 95% (compounds 3 and 4 represent pre-chondrochloren peaks). c, characterization of the decarboxylase CndG. Determination of CndG kinetics was performed using purified compound 4 as substrate. Each point represents the average of three measurements, and the error bars indicate the standard deviation. The data were fit to the Michaelis-Menten equation by nonlinear regression.