Role of Bacillus subtilis BacB in the synthesis of bacilysin

Abstract

Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (l-alanine-[2,3-epoxycyclohexano-4]-l-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of l-anticapsin. These data, however, were not consistent with the putative functional annotation for these proteins whereby BacA, a prephenate dehydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize l-anticapsin. Here we demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we show that BacB is an oxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to l-anticapsin. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvic acid. A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis.

FIGURE 1.

BacB in the context of the bac operon of B. subtilis. a, di-peptide antibiotic bacilysin has an N-terminal alanine and anticapsin at the C terminus. b, biosynthetic route for bacilysin production branches off the aromatic amino acid biosynthesis pathway at prephenate. c, BacB is a bicupin with two putative active sites, each containing a bound metal ion. d, topology diagram of BacB (23). e, electron density map of the active site of the N-terminal domain. The metal ion along with two water molecules is shown along with residue Lys-107 (at the entrance of the β barrel, shown to be crucial for catalytic activity), and residues His-50, His-52, His-91, and Gln-56 (coordinating the metal ion). f, (F_o − F_c) electron density map at the C-terminal domain could be interpreted as a bound PPY. The metal ion along with the coordinating residues His-162, His-164, His-202, and Gln-168 are shown.

FIGURE 2.

Experimental evidence for a bound phenylpyruvate at the C-terminal active site of BacB. a, DNPH assay with denatured BacB extract. (○), DNPH alone; (■), phenylalanine treated with DNPH (negative control); (▴), phenylpyruvic acid-sodium salt treated with DNPH (positive control); (●), BacB treated with DNPH. b, ESI-spectrum of BacB extract in positive as well as in negative ion modes shows the presence of phenylpyruvic acid.

FIGURE 3.

HPLC and UV spectrophotometric studies of the products of BacA and BacB enzymes. a, HPLC elution profiles of the BacA product with prephenate at different time points (1, 5, 10, and 60 min; 220 nm, 280 nm). b, HPLC elution profile of prephenate incubated with BacA alone, prephenate with BacB alone and prephenate with BacA and BacB. c, UV spectrophotometric time course measurement of the catalytic activity of BacA on prephenate. These spectra were recorded at intervals of 30 s. d, UV spectrophotometric time course measurement of the catalytic activity of BacB. These spectra were recorded at intervals of 30 s.

FIGURE 4.

Auto-oxidation of the product of BacA. a, product obtained by the activity of BacA on prephenate is prone to oxidation upon prolonged incubation. The spectroscopic profile of this product resembles that of BacB. Here, (■), prephenate alone incubated for 12 h; (▴), BacA with prephenate incubated for 12 h; (●), BacA and BacB with prephenate incubated for 15 min. b, reference UV spectra of potential substrates and products of the Bac proteins. (■), 4-hydroxy phenylpyruvic acid, (⊗), l-phenylalanine; (○), dl-tryptophan; (●), prephenate; (◊), l-tyrosine, (★), phenylpyruvic acid.

FIGURE 5.

¹H NMR spectrometric studies. a, overlay of ¹H NMR spectra corresponding to prephenate alone, BacA incubated with prephenate, and BacA and BacB incubated with prephenate are shown. These spectra show the product formation through chemical shifts peak 1 (around 3.0 ppm) upon incubating prephenate with BacA and peak 2 (around 6.28–6.34 ppm) upon incubating prephenate with both BacA and BacB. These chemical shifts match the predicted ¹H NMR spectra of the products (also see supplemental Fig. S1). The details of sample preparation and data acquisition are discussed under “Experimental Procedures.” Approximately 70% pure barium prephenate (Sigma-Aldrich) was dissolved in 5 mm Tris-HCl buffer, pH 8.1, for the activity assays. b, time course recording of ¹H NMR spectra shows an increase in the product formation (6.28–6.34 ppm) upon incubating prephenate with both BacA and BacB. c, magnified ¹H NMR spectrum of the product (6.28–6.34 ppm) by the action of BacA and BacB enzymes upon incubation with prephenate for 45 min.

FIGURE 6.

Schematic of the reactions catalyzed by BacA and BacB. Prephenate is converted into an unstable intermediate 3-((1r-4r)-4-hydroxycyclohexa-2,5-dienyl)-2-oxopropanoic acid after a decarboxylation reaction catalyzed by BacA. BacB catalyzes the oxidation of this product to yield 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid. The synthesis of l-anticapsin from 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid would further require a ring epoxidation and a transamination reaction in the ketone group.

FIGURE 7.

Mass spectra of the reaction products of BacA and BacB. a, ESI spectra of BacA incubated with prephenate in the positive ionization mode. b, ESI spectra of the product formed by the incubation of BacA and BacB with prephenate in positive ionization mode. c, ESI spectra of the product formed by the incubation of BacA and BacB with prephenate in and in negative ionization mode. The mass of barium prephenate is 361.49 Da. The deconvoluted mass obtained by ESI-MS is 226.9 Da in the positive ion mode, which corresponds to the mass of prephenate without barium (calculated mass 226.04 Da). Incubation of BacA with prephenate results in products with masses of 181.9, 182.9, and 183.9 Da in the positive ionization mode (isotopic splitting). The difference in mass between the substrate and the product (45 Da) confirms the decarboxylation reaction catalyzed by BacA. The incubation of BacA and BacB with prephenate results in products with masses 182.9 (183.9 Da) in the positive ion mode and 180.9 (181.9 Da) in the negative ionization mode (with isotopic splitting).

FIGURE 8.

The catalytic activity of BacB and K107A variant. a, Michaelis-Menten plot for the activity of the native BacB. The kinetic parameters are as noted in the text. b, N-terminal domain of BacB participates in catalysis. (●), product formed by the incubation of BacA and BacB with prephenate for 15 min (λ_max, 293 nm); (■), incubation of BacA and the K107A mutant of BacB with prephenate for 15 min.