The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S, 3R)-diaminobutyrate production

Abstract

Non-ribosomal peptide synthetases (NRPSs) assemble metabolites of medicinal and commercial value. Both serine and threonine figure prominently in these processes and separately can be converted to the additional NRPS building blocks 2,3-diaminopropionate (Dap) and 2,3-diaminobutyrate (Dab). Here we bring extensive bioinformatics, in vivo and in vitro experimentation to compose a unified view of the biosynthesis of these widely distributed non-canonical amino acids that both derive by pyridoxal-mediated β-elimination of the activated O-phosphorylated substrates followed by β-addition of an amine donor. By examining monobactam biosynthesis in Pseudomonas and in Burkholderia species where it is silent, we show that (2S,3R)-Dab synthesis depends on an l-threonine kinase (DabA), a β-replacement reaction with l-aspartate (DabB) and an argininosuccinate lyase-like protein (DabC). The growing clinical importance of monobactams to both withstand Ambler Class B metallo-β-lactamases and retain their antibiotic activity make reprogrammed precursor and NRPS synthesis of modified monobactams a feasible and attractive goal.

Keywords: Chemistry.

Graphical abstract

Figure 1

Analysis of BGCs containing Dap and Dab operons (A) sulfazecin-bulgecin and the cryptic monobactam in B. thailandensis E264. sulG-sulH and dabA-dabB-dabC are operons responsible for the biosynthesis of l-Dap (3) and (2S,3R)-Dab (4), respectively. (B) Structures of MM42842, sulfazecin, and aztreonam.

Figure 2

Substrate specificity analysis of E264 A3T3 di-domain (A) A-domain substrate specificity in vitro assays of SulM A3T3 (green bars) and E264 A3T3 (red bars). The purified proteins of SulM A3T3 and E264 A3T3 are also shown. (B) Amino acid substrates used in A-domain assays.

Figure 3

Nitrocefin assays, bioassays, and UPLC-HRMS assays of P. acidophila ΔsulG genetic and chemical complementation (A) P. acidophila ΔsulG complemented by sulGH or l-Dap (3). (B) P. acidophila ΔsulG complemented by dabABC or (2S,3R)-Dab (4). (a). Nitrocefin assay. (b). Bioassay on E. coli ESS.

Figure 4

In vivo characterization of DabA and DabC (A) Motifs I-III conserved in DabA and GHMP family kinases and Motif IV conserved in Pdux, BluE, and other l-Thr kinases. Identical and similar residues are highlighted in blue and yellow, respectively. Asterisks (∗) indicate residues demonstrated to be critical to enzyme activity. (B) Nitrocefin assays, bioassays, and UPLC-HRMS assays in P. acidophila ΔsulG (pUCP20-ANT2-MCS/dabBC) showing sulfazecin (1) instead of MM42842 (5) was produced. (+) induced, (−) uninduced. (C) Nitrocefin assays, bioassays, and UPLC-HRMS assays of MM42842 (5) produced in P. acidophila ΔsulG (pUCP20-ANT2-MCS/dabC) in the presence of 2 mM ACPAA (17), ΔsulG supplemented with ACPAA (17), or induced dabC in ΔsulG (pUCP20-ANT2-MCS/dabC) omitting of ACPAA (17). (a). Nitrocefin assay. (b). Bioassay on E. coli ESS.

Figure 5

In vitro characterization of DabB (A) UV-vis spectra of DabB, complex of DabB with OPT (9), and DabB-OPT with l-Asp or l-Glu. The wavelength of maximum absorbance is labeled. (B) UPLC-HRMS assays of ACPAA (17) trimethyl ester produced from OPT and l-Asp by DabB. (+): full reaction. (−): control reaction without DabB. (C) UV-vis spectra of DabB, complex of DabB with OPS (6), and DabB-OPS with l-Asp or l-Glu. The wavelength of maximum absorbance is labeled. (D) UPLC-HRMS assays of ACEAA (16) trimethyl ester produced from OPS and l-Asp by DabB. (+): full reaction. (−): control reaction without DabB.

Figure 7

The proposed Dab and Dap biosynthetic pathways (A) Proposed biosynthetic pathway to (2S,3R)-Dab (4) by DabA, DabB, and DabC. (B) Proposed alternative biosynthetic pathway to l-Dap (3) by DabB and DabC.

Figure 6

Synthesis of ACPAA (17)