Gatekeeping versus promiscuity in the early stages of the andrimid biosynthetic assembly line

Abstract

The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal peptide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-beta-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-beta-Phe, which in turn is activated by AdmJ and ATP to form (S)-beta-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-beta-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or AdmI. AdmF will also use either stereoisomer of phenylalanine or beta-Phe when presented on AdmA and AdmI, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA.

Figure 1

Highly dissociated biosynthetic pathway of andrimid. The early biosynthetic steps are emphasized. The polyunsaturated acyl chain is installed onto the phosphopantetheinyl (Ppant) arm of holo-AdmA (HS-AdmA) through the actions of a type II PKS. The resulting octatrienoyl-S-AdmA (blue) serves as substrate for the self-acylating transglutaminase homologue AdmF (yellow). (S)-β-Phe is synthesized from l-Phe by the MIO-containing aminomutase AdmH and is activated to the corresponding AMP-ester by the A domain AdmJ. AdmJ then installs (S)-β-Phe onto the Ppant arm of holo-AdmI (HS-AdmI), leading to the formation of (S)-β-Phe-S-AdmI (red). AdmF catalyzes the formation of an isopeptide bond between the octatrienoyl chain and the amine group of α-phenylalanine through the acylation of its active site cysteine (Cys90). The hybrid biosynthetic pathway proceeds through six T domains before final tailoring leads to the maturation of the final andrimid antibiotic.

Figure 2

Early gatekeepers in the biosynthesis and loading of the phenylalanine building block. a) HPLC traces of the AdmH aminomutase reaction. The AdmH reaction was carried out for 90 min at 30 °C using [¹⁴C]-labeled l-Phe as substrate. Non-labeled cinnamic acid, (S)-β-Phe, and (R)-β-Phe and [¹⁴C]-labeled l-Phe were used as standards. The AdmH reaction and standards were analyzed by chiral HPLC using an Astec Chirobiotic T2 column and a 9:1 (methanol/water) solvent system. The AdmH reaction produced (S)-β-Phe and cinnamic acid. b) Adenylation of phenyla-nine isomers by AdmJ. ATP/pyrophosphate assay carried out using (S)-β-Phe, (R)-β-Phe, and l-Phe. c) AdmJ-catalyzed loading of (S)-β-Phe on AdmI and AdmA. [¹⁴C]-labeled (S)-β-Phe was incubated with holo-AdmA in the absence and presence of AdmJ (lanes 1 and 2, respectively). [¹⁴C]-labeled (S)-β-Phe was incubated with holo-AdmI in the absence and presence of AdmJ (lanes 4 and 5, respectively). All reactions were quenched after 60 min of incubation at 30 °C. Left, SDS–PAGE; right, autoradiogram.

Figure 3

FT mass spectrum of the reverse AdmF reaction. Shown are the 10+ charge state of AdmA and the 9+ charge state of AdmI. The butyryl chain and (S)-β-Phe were loaded onto the reciprocal thiolation domain AdmI (peak 2) and AdmA (peak 1), respectively. The AdmF reaction led to the formation of two products, holo-AdmI (peak 3) and butyryl-(S)-β-Phe-S-AdmA (peak 4). Product formation was confirmed by observation of the butyryl-(S)-β-Phe-S-pantetheine ejection product within 2 ppm mass accuracy.

Figure 4

Recognition of amine nucleophiles by AdmF. FT mass spectrum (10+ charge state) of the AdmF reaction products arising from the isopeptide formation between the acyl chain of butyryl-S-AdmA and aminoacyl-S-AdmI. AdmI was loaded with the amine donors (S)-β-Phe, (R)-β-Phe, d-Phe, l-Phe, and (S)-β-Ala (top to bottom). All reactions were quenched after 30 min of incubation at 30 °C.

Figure 5

AdmF accepts a wide array of acyl chain donors. FT mass spectra of the AdmF reaction products obtained using AdmA T domains loaded with the following acyl chain donors with (S)-β-Phe-S-AdmI.

Figure 6

AdmF accepts octatrienoyl-S-NAC as surrogate substrate. a) FT mass spectra of the free AdmF (yellow) using the octatrienoyl-S-NAC as substrate, leading to the formation of acylated-AdmF (blue). b) Octatrienoyl-S-NAC feeding of E. coli host cells expressing the admA gene knockout of the andrimid biosynthetic cluster. Liquid cultures of the cells mentioned above were grown in the presence of 200 µM octatrienoyl-S-NAC and incubated at 30 °C for 24 h. The resulting liquid medium was extracted with ethyl acetate and submitted to preparative HPLC (see Methods). Cells that were not fed the octatrienoyl-S-NAC did not produce any andrimid, as illustrated by the absence of a clearing zone of the bioassay plate and absence of a peak on the corresponding HPLC trace (see no. 1). Cells that were fed the octatrienoyl-S-NAC produced both antibiotic activity and a peak that coeluted with an authentic andrimid standard (see HPLC traces and bioassays 2 and 3, respectively). Notably, the octatrienoyl-S-NAC did not possess any apparent antibiotic activity (bioassay 4).