Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis

Abstract

Glycosylation is a common modification reaction in natural product biosynthesis and has been known to be a post-assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis, glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation and in vitro pathway reconstitution, we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-(3-aminophenyl)3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free β-ketoacid that subsequently undergoes decarboxylation.

Figure 1.. Proposed biosynthetic pathways to pactamycin.

a. Biosynthetic origin of pactamycin structural components; b. Previously proposed pathways to pactamycin; c. Biosynthetic gene cluster of pactamycin in Streptomyces pactum. Colored thick arrows represent genes that are directly involved in this study.

Figure 2.

In vivo evidence for the involvement of 3AP-3OP-ACP in pactamycin biosynthesis. Representative mass spectrum of a. the BuOH extract of ΔptmH showing the presence of TM-025; b. the BuOH extract of ΔptmH/ΔptmT showing the absent of TM-025; c. the BuOH extract of ΔptmH/ΔptmT complemented with 3AP-3OP-SNAC showing the recovery of TM-025 production; d. the EtOAc extract of ΔptmH showing the presence of TM-026; e. the EtOAc extract of ΔptmH/ΔptmT showing the absent of TM-026; f. the EtOAc extract of ΔptmH/ΔptmT complemented with 3AP-3OP-SNAC showing the recovery of TM-026 production; g. A newly proposed biosynthetic pathway to pactamycin showing that glycosylation occurs on an ACP-bound polyketide intermediate. Experiments 2a-f were performed independently three times with similar results.

Figure 3.

Loading of 3ABA to the acyl-carrier protein PtmI and formation of β-ketoacyl-PtmI. a. Conversion of apo-PtmI to holo-PtmI, loading of 3ABA or malonate to PtmI, and decarboxylative Claisen condensation of 3ABA-PtmI and malonyl-PtmI to give β-ketoacyl-PtmI; b. Deconvoluted mass spectrum of apo-PtmI; c. Deconvoluted mass spectrum of holo-PtmI; d. Deconvoluted mass spectrum of a reaction mixture containing holo-PtmI, PtmS, 3ABA, ATP and MgCl₂; e. Deconvoluted mass spectrum of holo-PtmI incubation with malonyl-CoA; f. Deconvoluted mass spectrum of PtmK incubation with 3ABA-PtmI and malonyl-PtmI. The gluconoylated proteins are labeled with “Gluc”. Decarboxylation of malonyl-PtmI (12480.00 Da) and its Gluc analogue (12658.40 Da) yielded acetyl-PtmI (12436.60 Da) and its Gluc analogue (12614.40 Da), respectively. Experiments 3b-f were performed independently three times with similar results.

Figure 4.

Biochemical characterization of PtmO. a. Incubation of 3AP-3OP-PtmI with recombinant PtmO resulted in 3AAP; b. HPLC chromatogram of enzymatic reaction with boiled PtmO; c. HPLC chromatogram of enzymatic reaction with 10 μM PtmO; d. HPLC chromatogram of enzymatic reaction with 5 μM PtmO; e. HPLC chromatogram of standard 3-aminoacetophenone (3AAP). HPLC chromatograms were recorded based on UV absorption at 254 nm. Experiments 4b-d were performed independently at least three times with similar results.

Figure 5.

In vitro reconstitution of the PKS and the glycosyltransferase proteins involved in pactamycin biosynthesis. a. Enzymatic reactions from 3ABA to GlcNac-3ABA-PtmI and to GlcNAc-β-ketoacyl-PtmI; b. Deconvoluted mass spectrum of a reaction mixture containing PtmS, PtmI, and PtmJ; c. Deconvoluted mass spectrum of a reaction mixture with PtmS, PtmI, PtmK, and PtmJ; d. Deconvoluted mass spectrum of a reaction mixture with PtmS, PtmI, PtmK, and PtmJ using 3ABA-2,4,5,6-d₄ as substrate; e. Deconvoluted mass spectrum of a reaction mixture with PtmS, PtmI, PtmK, and boiled PtmJ. Magenta circle, holo-PtmI; blue circle, acetyl-PtmI; black circle, malonyl-PtmI; red diamond, 3ABA-PtmI; purple triangle, 3AP-3OP-PtmI; orange star, GlcNAc-3ABA-PtmI; yellow star, GlcNAc-3AP-3OP-PtmI. The gluconoylated proteins are labeled with “Gluc”. Deuterated species are labeled with d₄. Experiments 5b-e were performed independently at least three times with similar results.

Figure 6.

Characterization of the substrate selectivity of PtmK. a. experimental design for PtmK selectivity using 3ABA-SNAC as substrate; b. mass spectrum of PtmJ reaction mixture showing that 3ABA-SNAC can be glycosylated to give GlcNAc-3ABA-SNAC; c. deconvoluted mass spectrum of PtmK reaction mixture showing that only β-ketoacyl-PtmI was produced. Experiments 6b-c were performed independently three times with similar results.