Investigation of anticapsin biosynthesis reveals a four-enzyme pathway to tetrahydrotyrosine in Bacillus subtilis.
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.
Mahlstedt
Sarah A
SA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
Christopher T
CT
D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D024322:Amino Acids, Aromatic / Q000096:biosynthesis / Q000737:chemistry; D000600:Amino Acids, Dicarboxylic / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry; D019748:Carbon-Carbon Double Bond Isomerases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002262:Carboxy-Lyases / Q000096:biosynthesis* / Q000737:chemistry; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry; D053138:Cyclohexenes / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000096:biosynthesis* / Q000737:chemistry; D011302:Prephenate Dehydratase / Q000096:biosynthesis; D015398:Signal Transduction / Q000502:physiology*; D000637:Transaminases / Q000096:biosynthesis* / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
D024322:Amino Acids, Aromatic; D000600:Amino Acids, Dicarboxylic; D001426:Bacterial Proteins; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D004151:Dipeptides; D014443:Tyrosine; C015478:pretyrosine; C000097:anticapsin; C006674:bacilysin; D044925:Oxidoreductases Acting on CH-CH Group Donors; D000637:Transaminases; D002262:Carboxy-Lyases; D011302:Prephenate Dehydratase; D019748:Carbon-Carbon Double Bond Isomerases; D000409:Alanine; C005550:prephenic acid