Publications

• Investigation of anticapsin biosynthesis reveals a four-enzyme pathway to tetrahydrotyrosine in Bacillus subtilis.

  Mahlstedt S.A., Walsh C.T.

  Bacillus subtilis produces the antibiotic anticapsin as an L-Ala-L-anticapsin dipeptide precursor known as bacilysin, whose synthesis is encoded by the bacA-D genes and the adjacent ywfGH genes. To evaluate the biosynthesis of the epoxycyclohexanone amino acid anticapsin from the primary metabolit ... >> More

  Bacillus subtilis produces the antibiotic anticapsin as an L-Ala-L-anticapsin dipeptide precursor known as bacilysin, whose synthesis is encoded by the bacA-D genes and the adjacent ywfGH genes. To evaluate the biosynthesis of the epoxycyclohexanone amino acid anticapsin from the primary metabolite prephenate, we have overproduced, purified, and characterized the activity of the BacA, BacB, YwfH, and YwfG proteins. BacA is an unusual prephenate decarboxylase that avoids the typical aromatization of the cyclohexadienol ring by protonating C(8) to produce an isomerized structure. BacB then catalyzes an allylic isomerization, generating a conjugated dienone with a 295 nm chromophore. Both the BacA and BacB products are regioisomers of H(2)HPP (dihydro-4-hydroxyphenylpyruvate). The BacB product is then a substrate for the short chain reductase YwfH which catalyzes the conjugate addition of hydride at the C(4) olefinic terminus using NADH to yield the cyclohexenol-containing tetrahydro-4-hydroxyphenylpyruvate H(4)HPP. In turn, this keto acid is a substrate for YwfG, which promotes transamination (with L-Phe as amino donor), to form tetrahydrotyrosine (H(4)Tyr). Thus BacA, BacB, YwfH, and YwfG act in sequence in a four enzyme pathway to make H(4)Tyr, which has not previously been identified in B. subtilis but is a recognized building block in cyanobacterial nonribosomal peptides such as micropeptins and aeruginopeptins. << Less

  Biochemistry 49:912-923(2010) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Role of Bacillus subtilis BacB in the synthesis of bacilysin.

  Rajavel M., Mitra A., Gopal B.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 284:31882-31892(2009) [PubMed] [EuropePMC]

• Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin.

  Parker J.B., Walsh C.T.

  Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven Bacillus subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have pr ... >> More

  Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven Bacillus subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have previously noted the formation of two diastereomers of tetrahydrotyrosine (4S- and 4R-H(4)Tyr) by tandem action of the four purified enzymes BacABGF. BacC (oxidase) and BacD (ligase) have been hypothesized to be remaining late stage enzymes in bacilysin biosynthesis. Using a combination of BacCD in vitro studies, B. subtilis deletion mutants, and isotopic feeding studies, we were able to determine that the H(4)Tyr diastereomers are actually shunt products that are not on-pathway to bacilysin biosynthesis. Dihydroanticapsin and dihydrobacilysin accumulate in extracts of a ΔbacC strain and are processed to anticapsin and then bacilysin upon addition of BacC and BacD, respectively. These results suggest the epoxide group in bacilysin is installed in an earlier step of bacilysin biosynthesis, while BacC oxidation of the C(7)-hydroxyl and the subsequent BacD ligation of anticapsin to l-Ala are the penultimate and ultimate steps of bacilysin biosynthesis, respectively. << Less

  Biochemistry 52:889-901(2013) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Olefin isomerization regiochemistries during tandem action of BacA and BacB on prephenate in bacilysin biosynthesis.

  Parker J.B., Walsh C.T.

  BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H(2)HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, conve ... >> More

  BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H(2)HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, converting the 1,4-diene in prephenate to the endocyclic 1,3-diene in Δ(4),Δ(8)-dihydrohydroxyphenylpyruvate (en-H(2)HPP). BacB then performs an allylic isomerization to bring the diene into conjugation with the 2-ketone in the product Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP). To prove that BacA acts regiospecifically on one of the two prochiral olefins in prephenate, we generated 1,5,8-[(13)C]-chorismate from bacterial fermentation of 5-[(13)C]-glucose and in turn produced 2,4,6-[(13)C]-prephenate via chorismate mutase. Tandem action of BacA and BacB gave 2,4,8-[(13)C]-7R-ex-H(2)HPP, showing that BacA isomerizes only the pro-R double bond in prephenate. Nonenzymatic isomerization of the BacA product into conjugation gives only the Δ(3)E-geometric isomer of Δ(3),Δ(5)-ex-H(2)HPP. On the other hand, acceleration of the allylic isomerization by BacB gives a mixture of the E- and Z-geometric isomers of the 7R-product, indicating some rerouting of the flux, likely through dienolate geometric isomers. << Less

  Biochemistry 51:3241-3251(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.