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, 76 (2), 150-6

The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

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The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

Akshaya Ravichandran et al. J Nat Prod.

Abstract

Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by the Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinates the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides.

Figures

Figure 1
Figure 1
Covalent structure of occidiofungin. (R1 = H or OH; R2 = H or Cl)
Figure 2
Figure 2
RP-HPLC Chromatograms. A. Chromatogram of the final purification step of the wild-type occidiofungin fraction at 220 nm using a 4.6 × 250 mm C18 column. B. Chromatogram of the final purification step of ocfN mutant occidiofungin fraction at 220 nm using a 4.6 × 250 mm C18 column. C. Overlay of the wild-type (black) and the mutant (grey) fractions of occidiofungin.
Figure 3
Figure 3
TOCSY (left panel) and HSQC (right panel) spectra of BHY4 in the wild-type sample. The proportions of Asn1 and BHN1 variants were determined by the measurement of the Ha-Ca cross peak intensities of BHY4 in the HSQC spectra. These values are listed next to their corresponding peaks in the right panel. The peaks in red and green represent the BHY4 peaks associated with BHN1 and Asn1 variants, respectively. Based on the calculation of their relative proportions, i.e. (34.81 + 87.97 for the BHY4 peaks found in the BHN1 conformational variants) and (32.79 + 37.61 for the BHY4 peaks found in the Asn1 conformational variants), the approximate proportion of the Asn1 variants could be calculated as (32.79 + 37.61)/(34.81 + 87.97) + (32.79 + 37.61).
Figure 4
Figure 4
ESI mass spectrometry. A. ESI-MS data of purified wild-type occidiofungin fraction. B. ESI-MS data of purified ocfN mutant occidiofungin fraction.
Figure 5
Figure 5
TOCSY fingerprint region (NH correlations). A. NH correlations in the wild-type sample. The two BHN1 and four Asn1 spin systems present in the wild-type sample are colored red. B. NH correlations in the ocfN mutant sample. C. Overlay of the NH correlations found in the wild-type and ocfN mutant samples. NH correlations that are not present in the ocfN mutant sample are colored green.
Figure 6
Figure 6
Schematic of occidiofungin ring closure. The completely synthesized eight amino acid linear peptide is bound by a 4-phosphopantetheine cofactor (ppan) linker to the thiolation (T) domain. The peptide varies by the presence or absence of a hydroxy group on the β-carbon of Asn1. The TE domain of OcfD is capable of forming the cyclic peptide of both variants in the absence of a functional OcfN cyclase thioesterase. However, it is not as efficient at producing the Asn1 cyclic peptide variant as OcfN. In the presence of a functional OcfN cyclase thioesterase, novel diastereomers of occidiofungin are formed by the selective ring closure of the Asn1 cyclic peptide. R1 and R2 are BHN1 and Asn1, respectively.
Figure 7
Figure 7
Comparison of the bioactivity from the wild-type and ocfN mutant occidiofungin fractions. A. MICs of wild-type and ocfN mutant fraction determined by CLSI M27-A3 method in RPMI 1640. B. Comparison of the CFUs in the MIC wells of wild-type fraction to the corresponding well having the same concentration of the ocfN mutant occidiofungin fraction. Asterisks represent no detectable colonies in the MIC wells of the wild-type occidiofungin fraction. Black and grey bars are ocfN mutant and wild-type fractions, respectively. Standard deviations for the CFU measurements are presented.
Figure 8
Figure 8
Potato dextrose agar plates were inoculated with each of the strains and incubated for 3 days at 28°C. The plates were oversprayed with the indicator fungus Geotrichum candidum and incubated overnight. A: The wild-type strain MS14; B: Negative control MS14GG78 (ocfJ::nptII); C: MS14GG88 (ocfN::nptII).

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