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 ...

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B 12 -dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central ␣/␤-core G domain that is typical of the GTPase family, as well as ␣-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP-and GTP-bound forms of this protein.Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.In the past few decades, an increasing number of guanine nucleotide-binding protein (G proteins) that act as chaperones in the assembly of target metalloenzymes have been described (1). These include UreG (2, 3), HypB (4, 5), CooC (6), and MeaB (7), which are involved in the metallocenter assembly of urease, NiFe-hydrogenase, CO dehydrogenase, and B 12 -dependent methylmalonyl-CoA mutase, respectively. Typically, G proteins act as molecular switches, with regions known as switch I and switch II undergoing large conformational changes upon GTP hydrolysis to communicate a signal. Although members of this metallochaperone G protein subfamily (called the G3E family) share appropriate sequence motifs (8) and exhibit low GTPase activity (3,4,6,9), their exact function with respect to target metalloenzymes remains to be determined. MeaB itself differs from the other G3E G proteins in that it possesses N-and C-terminal extensions of unknown function.MeaB is an auxiliary protein associated with methylmalonylCoA mutase (MCM) 2 (7, 9, 10), a coenzyme B 12 (adenosylcobalamin)-dependent enzyme that catalyzes the chemically challenging 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA using radical-based chemistry (11,12). A human orthologue of MeaB, MMAA, has been found to be the locus of mutations associated with type A (cblA) methylmalonic aciduria (MMA) (13), a rare congenital disease that manifests itself d...

Prephenate is the direct precursor of phenylpyruvate and 4-hydroxyphenylpyruvate in the biogenesis of phenylalanine and tyrosine by action of the decarboxylative, aromatizing enzymes prephenate dehydratase and dehydrogenase, respectively. The recent characterization of BacA in bacilysin biosynthesis as a non-aromatizing decarboxylase reveals a new route from prephenate in the biosynthesis of nonproteinogenic amino acids. This study describes two additional enzymes, AerD from Planktothrix agardhii and SalX from Salinispora tropica, that utilize the central building block prephenate for flux down distinct pathways to amino acid products, representing a new metabolic fate for prephenate and establishing a new family of non-aromatizing prephenate decarboxylases.Prephenate has been known for decades as the product of the celebrated 3,3-sigmatropic rearrangement of chorismate, a reaction that is classically followed by formation of the aromatic amino acids phenylalanine and tyrosine (1). Prephenate dehydratase and prephenate dehydrogenase are the enzymes that direct prephenate to Phe and Tyr by decarboxylation and aromatization of the cyclohexenyl ring with concomitant loss of the C-7 hydroxide or hydride, respectively. Recently we characterized the Bacillus subtilis prephenate dehydratase homolog BacA, which is involved in the biosynthesis of bacilysin (1, Figure 1), and catalyzes the first step of a 4-enzyme pathway that transforms prephenate to a distinct non-aromatic amino acid product (2). Like prephenate dehydratase, BacA decarboxylates prephenate at C-4 yet uniquely protonates C-6 or C-6' to yield the endocyclic diene dihydro-4-hydroxyphenylpyruvate (H 2 HPP, 5, Figure 2A) (2). This diene is then subject to nonenzymatic isomerization to the exocyclic diene in a reaction that can be dramatically accelerated by BacB to yield the indicated H 2 HPP regioisomer 6. Here we identify two homologs to BacA from distinct microbial secondary metabolite pathways that catalyze the same non-aromatizing decarboxylation of prephenate, thus establishing a new family of enzymes. The first of these is AerD from the cyanobacterium Planktothrix agardhii, which is involved in the production of the nonribosomal glycopeptides

Summary 2,5-Dihydrophenylalanine (H2Phe) is a multi-potent non-proteinogenic amino acid produced by various Actinobacteria and Gammaproteobacteria. While the metabolite was discovered over forty years ago, details of its biosynthesis have remained largely unknown. We show here that L-H2Phe is a secreted metabolite in Photorhabdus luminescens cultures and a precursor of a recently described 2,5-dihydrostilbene. Bioinformatic analysis suggested a candidate gene cluster for the processing of prephenate to H2Phe, and gene knockouts validated that three adjacent genes plu3042-3044 were required for H2Phe production. Biochemical experiments validated Plu3043 as a non-aromatizing prephenate decarboxylase generating an endocyclic dihydro-hydyroxyphenylpyruvate. Plu3042 acted next to transaminate the Plu3043 product, precluding spontaneous exocyclic double bond isomerization and yielding 2,5-dihydrotyrosine. The enzymatic products most plausibly on path to H2Phe illustrate the versatile metabolic rerouting of prephenate from aromatic amino acid synthesis to antibiotic synthesis.

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