Investigation of Anticapsin Biosynthesis Reveals a Four-Enzyme Pathway to Tetrahydrotyrosine in Bacillus subtilis

Abstract

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.Bacillus subtilis produces a variety of polyketide and peptide-derived antibiotics, such as difficidin, bacillaene, mycosubtilin, fengycin, and surfactin (1). Bacilysin (1), a deceptively simple example of a B. subtilis antibiotic (Figure 1a), was first isolated in 1946 (2). Its structure was solved in 1970 (3), though corrections to its assigned stereochemistry were made decades later (4,5). Over the years this compound has been referred to by a variety of names, including bacillin and tetaine (6,7), and has been identified in other Bacillus species (8-10). Bacilysin is a dipeptide consisting of an N-terminal alanine residue linked to a non-proteinogenic epoxycyclohexanone-containing amino acid referred to as anticapsin (2). This unusual residue is the key to bacilysin's antibiotic and antifungal activity (11). The dipeptide is exported by producing cells and can be taken up by a competitor via di-to oligopeptide uptake systems (12,13). Cytoplasmic peptidases cleave the dipeptide, releasing the anticapsin warhead (11,14). Anticapsin can then bind to the active site of the cell wall biosynthetic enzyme glucosamine-6-phosphate synthase as a mimic of the natural glutamine substrate, resulting in irreversible inhibition of the enzyme. Covalent attachment of anticapsin presumably arises from the reaction of an active site cysteine thiol with the epoxide functional group (14,15).The biosynthesis of anticapsin by B. subtilis has remained a mystery in the six decades since its isolation. Originally it was suspected that the amino acid was derived from either tyrosine † This work was supported in part by the ...

Two-Component Response Regulator DegU Controls the Expression of Bacilysin in Plant-Growth-Promoting Bacterium Bacillus amyloliquefaciens FZB42

“…The mode of antimicrobial activity is mainly due to the inhibition of glucosamine synthesis, leading to defects in the microbial cell wall [Kenig et al, 1976;Walton and Rickes, 1962]. It has been well established that prephenate, an intermediate of the aromatic amino acid biosynthetic pathway, is the primary metabolic precursor of the anticapsin moiety [Mahlstedt and Walsh, 2010;Roscoe and Abraham, 1966]. Anticapsin is released from bacilysin upon transport into the cell, making the essence of bacilysin antimicrobial activity [Perry and Abraham, 1979].…”

“…The ywfH gene product is involved in converting H 2 HPP 5 to H 4 HPP 6 , making the precursor for anticapsin; the function of bacC is unclear. The bacD and bacE gene products direct amino acid ligation and host bacterium self-protection, respectively [Mahlstedt and Walsh, 2010]. The bacABCDE gene cluster in FZB42 is collinear to B. subtilis 168, and its homology ranges between 84 and 93% [Chen et al, 2009a].…”

Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis

“…34−39 BtrR, a dual functional aminotransferase for the biosynthesis of 2-DOS in butirosin, has also been biochemically and structurally characterized. 40,41 Surprisingly, limited examples were available on the biochemical and structural characterization of aminotransferases responsible for scaffold construction/modification in the biosynthesis of bacterial secondary metabolites, including OxyQ (anhydrotetracycline, in vivo studies), 42 Neo-18 (neomycin) and BtrB (butirosin), 43 PvdH (pyoverdinesiderophore), 44 YwfG (anticapsin), 45 PctV (pactamycin), 46,47 and GenS2 (gentamycin). 48 The crystal structure of PigE, 49 an aminotransferase in prodigiosin biosynthesis, 50 was recently reported.…”

Biochemistry, genetics and regulation of bacilysin biosynthesis and its significance more than an antibiotic

“…The most crucial reports resolved the sequential action of BacABGF (four-enzyme pathway) to convert prephenate to 2S, 4S, 7R-and 2S,4R,7R-tetrahydrotyrosine (H 4 Tyr) diastereomers on the way to anticapsin [35][36][37]. To our general knowledge, BacA is responsible for decarboxylation of prephenate and specific isomerization of the pro-R double bond yields the endocyclic dienyl product 7R-endocyclic-dihydrohydroxyphenylpyruvate (H 2 HPP) [38].…”