Epimerization and substrate gating by a TE domain in β-lactam antibiotic biosynthesis

Abstract

Nonribosomal peptide synthetases are versatile engines of bioactive natural product biosynthesis that function according to the multiple carrier thiotemplate mechanism. C-terminal thioesterase (TE) domains of these giant modular proteins typically catalyze product release by hydrolysis or macrocyclization. We now report an unprecedented, dual-function TE that is involved in the biosynthesis of nocardicin A, which is the paradigm monocyclic β-lactam antibiotic. Contrary to our expectation, a stereodefined series of potential peptide substrates for the nocardicin TE domain failed to undergo hydrolysis. The stringent discrimination against peptide intermediates was overcome by prior monocyclic β-lactam formation at an L-seryl site. Kinetic data are interpreted such that the TE domain acts as a gatekeeper to hold the assembling peptide on an upstream domain until β-lactam formation takes place and then rapidly catalyzes epimerization, which has not been observed previously as a TE catalytic function, and thioesterase cleavage to discharge a fully fledged pentapeptide β-lactam harboring nocardicin G, the universal precursor of the nocardicins.

Figure 1. Isopenicillin N and Nocardicin A biosyntheses

(a) Isopenicillin N biosynthesis from NRPS ACV synthetase and isopenicillin N synthase. (b) Overall biosynthesis of nocardicin A from early stage peptide assembly initiated by pHPG biosynthesis to late stage nocardicin G modification. (c) Domain and module architecture of NRPSs NocA and NocB and predicted peptide products.

Figure 2. Substrates synthesized to probe the specificity of NocTE

Substrates vary in peptide length, C-terminal pHPG stereochemistry and O-seryl modification.

Figure 3. In vitro substrate profiling of NocTE

(a) Schematic of NocTE reactivity towards various substrates. Top: Linear and activated penta- and tri- peptides are poor substrates for NocTE. Lower: Pre-formation of β-lactam at the seryl position results in NocTE epimerization and hydrolysis activity. (b) HPLC analysis of β-lactam-containing thioester substrates with excised NocTE. All of the β-lactam-containing substrates were processed by NocTE and only one product was formed, containing a C-terminal D-pHPG. Top traces show NocTE incubated with 8a and 8b, forming 1 and bottom traces show NocTE incubated with 9a and 9b, forming 14.

Figure 4. ¹H-NMR spectrometric analysis of NocTE with epi-nocardicin G/nocardicin G-SNAC

(a) ¹H-NMR experiment demonstrating NocTE turnover of epi-nocardicin G/nocardicin G-SNAC. The bottom spectrum labeled “-NocTE” shows the substrates 11a/b before NocTE addition. “D” and “L” correspond to the N-acetyl peaks of SNAC of the nocardicin G-SNAC (11b) and epi-nocardicin G-SNAC (11a) thioesters, respectively. Arrows highlight the two C-4 diastereotopic hydrogen resonances (H-4α/β) of 1, whose appearance was monitored throughout the experiment. (b) ¹H-NMR comparison of synthetic epi-nocardicin G and nocardicin G with isolated deutero-nocardicin G from NocTE conversion of substrate 11a/b in D₂O.

Figure 5. Kinetic analysis and determination of NocTE as a dual epimerase/hydrolase

(a) Depiction of NocTE catalyzed conversion of epi-nocardicin G-SNAC (11a) and nocardicin G-SNAC (11b) to nocardicin G (1). (b) Plots of initial velocities vs. 11a and 11b concentration. Experiments were conducted in triplicate; data represents mean values ± standard deviation. (c) HPLC analysis of time points in competition experiments between 11b and 9b in the presence of NocTE, showing the appearance of 1 and 14.

Figure 6. Possible pathways to monocyclic β-lactam formation

Top, pathway represents in cis β-lactam formation, invoking an external enzyme responsible for cyclodehydration. Bottom, pathway represents in trans β-lactam formation catalyzed by a domain within the NocNRPS. Upon β-lactam formation NocTE catalyzes C-terminal epimerization and hydrolysis of 14.