Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line

Abstract

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,β-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

Fig. 1.

The tetrahydroisoquinoline family natural products. (A) Isotope feeding experiment showed that l-tyrosine, l-methionine, glycine, and d,l-ornithine could be incorporated into NDM. (B) Structures of QNC and ET-743.

Fig. 2.

Proposed mechanism of the TKase-ACP systems involved in the tetrahydroisoquinoline biosynthesis by diverting a hydroxyacyl unit from ketosugars into a nonribosomal peptide assembly line. (A) Comparison between the TKase-ACP systems in NDM and QNC biosynthesis. aa, amino acid; I/S, identity/similarity. (B) The proposed biosynthetic pathway and catalytic cycle by TKase-ACP system. (C) The ThDP-dependent reaction mechanism catalyzed by TKase.

Fig. 3.

Verification of the necessary role of TKase in the biosynthesis of the tetrahydroisoquinoline family. (A) HPLC analysis (UV at 270 nm). I, WT S. lusitanus NRRL 8034. II, mutant S. lusitanus TG3001 (ΔnapB); III, mutant S. lusitanus TG3003 (TG3001 harboring the napB–napC–napD expression plasmid pTG1012); IV, mutant S. lusitanus TG3005 (TG3001 harboring the qncN–qncM–qncL expression plasmid pTG1013). (B) LC-MS analysis. I, WT S. lusitanus NRRL 8034; II, mutant S. lusitanus TG3002 (ΔnapD); III, mutant S. lusitanus TG3004 (TG3002 harboring the napB–napC–napD expression plasmid pTG1012); IV, mutant S. lusitanus TG3006 (TG3002 harboring the qncN–qncM–qncL expression plasmid pTG1013). ▼, NDM.

Fig. 4.

Investigation of the possible precursors of C9–C9′ by feeding experiments with ¹³C-labeled sugars. (A) ¹³C NMR spectra of CN-NDM without feeding (I), with feeding of [2-¹³C]glucose (II), and with feeding of [1-¹³C]fructose (III). (B) Proposed mechanism of relative sugar metabolism and incorporation into NDM. Fru, fructose; Glu, glucose; G-6-P, glucose 6-phosphate; G6P-L, 6-phosphoglucono-δ-lactone; G6P-A, 6-phosphogluconate; R-5-P, ribulose-5- phosphate.

Fig. 5.

Biochemical characterization of the TKase-ACP systems in vitro. (A) HPLC analysis of enzymatic reaction. ○, apo-ACP; ●, holo-ACP; ▼, glycolicacyl-S-ACP; ◆, glycolicacyl-O-glycolicacyl-S-ACP; ▽, QncN or QncL; ◇, PDH E3. (B) Time course of enzymatic reaction using X-5-P (I), F-6-P (II), S-7-P (III), HPA (IV), and DHA (V) as donor substrates, respectively. The concentration of substrate used in the assay is 10 mM for DHA and 2 mM for others. (C) Q-TOF-MS analysis of holo-ACP (I), glycolicacyl-S-ACP (II), and glycolicacyl-O-glycolicacyl-S-ACP (III) using X-5-P as substrate. (D) Q-TOF-MS analysis of holo-ACP (I), glycolicacyl-S-ACP (II), and glycolicacyl-O-glycolicacyl-S-ACP (III) using [2-¹³C]F-6-P as substrate. (E) Proposed mechanism of the in vitro enzymatic reaction catalyzed by the TKase-ACP system.

Fig. 6.

Summary of the origins of the two-carbon unit in the biosynthesis. (A) YerE catalyzed the attachment of the C₂ branched chain onto sugar from pyruvate by a ThDP-dependent decarboxylation process. (B) A hydroxymalonyl-ACP derived from the glycolytic pathway as an extender unit for PKS involved in the biosynthesis of zwittermicin. (C) A unique NRPS extender unit derived from ketose phosphates through (α,β-dihydroxyethyl)-ThDP and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis.