bacilysin and prephenic-acid

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.

Topics: Alkenes; Bacterial Proteins; Chorismate Mutase; Cyclohexanecarboxylic Acids; Cyclohexenes; Dipeptides; Isomerism

The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H(4)Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Δ(4),Δ(8)-7R-dihydrohydroxyphenylpyruvate (en-H(2)HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C(4) to yield tetrahydrohydroxyphenylpyruvate (H(4)HPP), a transamination away (via BacF) from 2S-H(4)Tyr. Incubations of the pathway enzymes in D(2)O yield deuterium incorporation at C(8) from BacA and then C(9) from BacB action. By (1)H NMR analysis of samples of H(4)Tyr, the stereochemistry at C(4), C(8), and C(9) can be assigned. BacG (followed by BacF) converts 3E-ex-H(2)HPP to 2S,4R,7R-H(4)Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C(4), 2S,4S,7R-H(4)Tyr. Given that bacilysin has the 2S,4S stereochemistry in its anticapsin moiety, it is likely that the 2S,4S-H(4)Tyr is the diastereomer "on pathway". NMR determination of the stereochemistry of the CHD samples at C(8) and C(9) allows assignment of all stereogenic centers (except C(3)) in this unusual tetrahydro-aromatic amino acid building block, giving insights into and constraints on the BacA, BacB, and BacG mechanisms.

Topics: Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Carbon-Carbon Double Bond Isomerases; Cyclohexanecarboxylic Acids; Cyclohexenes; Dipeptides; Nuclear Magnetic Resonance, Biomolecular; Oxidoreductases Acting on CH-CH Group Donors; Prephenate Dehydratase; Stereoisomerism; Transaminases; Tyrosine

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.

Topics: Alanine; Amino Acids, Aromatic; Amino Acids, Dicarboxylic; Bacillus subtilis; Bacterial Proteins; Carbon-Carbon Double Bond Isomerases; Carboxy-Lyases; Cyclohexanecarboxylic Acids; Cyclohexenes; Dipeptides; Oxidoreductases Acting on CH-CH Group Donors; Prephenate Dehydratase; Signal Transduction; Transaminases; Tyrosine

Previously characterized mutants of Bacillus subtilis 168 were analyzed for the accumulation of the antibiotic bacilysin in culture broths and for the presence of bacilysin synthetase in cell extracts. All aro mutants tested were deficient in bacilysin biosynthesis but had synthetase activity. Mutants with lesions in tyrA and pheA produced normal levels of bacilysin, suggesting that prephenate is the primary metabolic precursor of bacilysin.

Topics: Anti-Bacterial Agents; Bacillus subtilis; Chemical Phenomena; Chemistry; Cyclohexanecarboxylic Acids; Cyclohexenes; Dipeptides; Mutation; Peptide Synthases