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Biosynthetic Study of Ustiloxin B, a Ribosomally Synthesized and Post-translationally Modified Peptide in Filamentous Fungi
叶 英南 篤志五十嵐 祐也尾崎 太郎梅村 舞子町田 雅之五味 勝也及川 英秋
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Biosynthetic Study of Ustiloxin B, a Ribosomally Synthesized and Post-translationally Modified Peptide in Filamentous Fungi

 Ustiloxins represented by ustiloxin B (1) were toxic fungal ribosomal peptides, isolated from rice false smut caused by the pathogenic fungus Ustilaginoidea virens (Figure 1).1-3These family members, including a structurally related phomopsin and asperipin-2a (Figure 1), exhibit potent antimitotic activity and inhibit microtubule assembly. Of particular structural interest is a maclocyclic structure constructed by an oxidative cyclization between the amino acid side chains. Recently, a biosynthetic gene cluster (ust) of 1was identified in the genome of Aspergillus flavus using a sequence motif-independent de novo detection algorithm (MIDDAS-M).4,5 The cluster contained a unique precursor protein, UstA, that has 16 repeated Tyl-Ala-Ile-Gly (YAIG) sequences. Recently, we elucidated the biosynthetic machinery of 1 through heterologous expression and in vitro studies.6 The results confirmed the oxidation enzymes harboring a DUF3328 motif for the macrocylization and unique modification enzymes for the amino acid-like side chain on the aromatic ring. In this presentation, we will discuss the biosynthesis of the first ribosomal peptide produced by filamentous fungi.

Proposed biosynthetic pathway of ustiloxins

 Previous gene knockout experiments showed eleven genes were responsible for the ustiloxin biosynthesis.7 LC-MS analysis of the extracts from five mutants, DustM, DustC, DustF1, DustF2, and DustD, showed the accumulation of ustiloxin derivatives (2-6). Except known ustiloxins, ustiloxin F (3) and ustiloxin C (6), our collaborators determined the structure of novel ustiloxins, N-desmethylustiloxin F (2), S-deoxyustiloxin H (4), and ustiloxin H (5).6These data enabled us to speculate the functions of the ustMCF1F2D genes. For the last transformation with UstD, condensation of a C3 nucleophile with an aldehyde form 8 of 6is more likely to be involved. Taken together, we proposed a biosynthetic pathway for 1 as shown in Scheme 1.6

Cyclic peptide formation catalyzed by unprecedented oxidation enzymes

 The gene knockout experiments of ustQYaYbindicated that these enzyme genes were involved in the characteristic cyclic peptide formation because the corresponding mutants completely abolished production of 2. The ustQ gene displays the highest similarity to a tyrosinase which usually catalyses an oxidation of tyrosine and dopamine. The ustYa and ustYb genes exhibit no homology with functionally characterized enzymes but have a common DUF3328 motif. For functional characterization of these genes, we conducted heterologous expression in Aspergillus oryzae.8We transformed the wild-type NSAR1 strain by applying the “tandem transformation8” strategy with different combination of plasmids, and three transformants, AO-ustAQYa, AO-ustAQYb, and AO-ustAQYaYb, were obtained (Figure 2). Although AO-ustAQYaand AO-ustAQYb produced no cyclic- and linear peptides, AO-ustAQYaYbgave 2 (Figure 2). Additional introduction of ustYb into AO-ustAQYa resulted in the production of 2 (Figure 2), suggesting that both UstY enzymes are responsible for the oxidative cyclization. The successful in vivo reconstitution of the biosynthetic machinery of 2further confirmed the importance of the three oxidation enzymes. Given the results of the gene inactivation studies, we speculated that the UstA protein is digested into 16 pieces of trideca-/tetradeca-peptides by intrinsic Kex2 prot

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 Ustiloxins represented by ustiloxin B (1) were toxic fungal ribosomal peptides, isolated from rice false smut caused by the pathogenic fungus Ustilaginoidea virens (Figure 1).1-3These family members, including a structurally related phomopsin and asperipin-2a (Figure 1), exhibit potent antimitotic activity and inhibit microtubule assembly. Of particular structural interest is a maclocyclic structure constructed by an oxidative cyclization between the amino acid side chains. Recently, a biosynthetic gene cluster (ust) of 1was identified in the genome of Aspergillus flavus using a sequence motif-independent de novo detection algorithm (MIDDAS-M).4,5 The cluster contained a unique precursor protein, UstA, that has 16 repeated Tyl-Ala-Ile-Gly (YAIG) sequences. Recently, we elucidated the biosynthetic machinery of 1 through heterologous expression and in vitro studies.6 The results confirmed the oxidation enzymes harboring a DUF3328 motif for the macrocylization and unique modification enzymes for the amino acid-like side chain on the aromatic ring. In this presentation, we will discuss the biosynthesis of the first ribosomal peptide produced by filamentous fungi.

Proposed biosynthetic pathway of ustiloxins

 Previous gene knockout experiments showed eleven genes were responsible for the ustiloxin biosynthesis.7 LC-MS analysis of the extracts from five mutants, DustM, DustC, DustF1, DustF2, and DustD, showed the accumulation of ustiloxin derivatives (2-6). Except known ustiloxins, ustiloxin F (3) and ustiloxin C (6), our collaborators determined the structure of novel ustiloxins, N-desmethylustiloxin F (2), S-deoxyustiloxin H (4), and ustiloxin H (5).6These data enabled us to speculate the functions of the ustMCF1F2D genes. For the last transformation with UstD, condensation of a C3 nucleophile with an aldehyde form 8 of 6is more likely to be involved. Taken together, we proposed a biosynthetic pathway for 1 as shown in Scheme 1.6

Cyclic peptide formation catalyzed by unprecedented oxidation enzymes

 The gene knockout experiments of ustQYaYbindicated that these enzyme genes were involved in the characteristic cyclic peptide formation because the corresponding mutants completely abolished production of 2. The ustQ gene displays the highest similarity to a tyrosinase which usually catalyses an oxidation of tyrosine and dopamine. The ustYa and ustYb genes exhibit no homology with functionally characterized enzymes but have a common DUF3328 motif. For functional characterization of these genes, we conducted heterologous expression in Aspergillus oryzae.8We transformed the wild-type NSAR1 strain by applying the “tandem transformation8” strategy with different combination of plasmids, and three transformants, AO-ustAQYa, AO-ustAQYb, and AO-ustAQYaYb, were obtained (Figure 2). Although AO-ustAQYaand AO-ustAQYb produced no cyclic- and linear peptides, AO-ustAQYaYbgave 2 (Figure 2). Additional introduction of ustYb into AO-ustAQYa resulted in the production of 2 (Figure 2), suggesting that both UstY enzymes are responsible for the oxidative cyclization. The successful in vivo reconstitution of the biosynthetic machinery of 2further confirmed the importance of the three oxidation enzymes. Given the results of the gene inactivation studies, we speculated that the UstA protein is digested into 16 pieces of trideca-/tetradeca-peptides by intrinsic Kex2 proteinases before the cyclization.9 To generate mature cyclic peptide 2, both the N- and C-terminal sequences of the tridecapeptide must be cleaved.

 With 2 producing AO-ustAQYaYb in hand, ustM was then incorporated into the transformant to create AO-ustAQYaYbM. However, the transformant produced a small amount of 3. Since DustT mutant accumulated 2, we postulated that UstT, a putative transporter, might improve the production. As expected, AO-ustAQYaYbMTtransformant resulted in a 3-fold production of 3, thus allowing us to propose the importance of the resistant gene for the biosynthesis of 3.

Plausible cyclization mechanism

 During the cyclic ether formation from a linear peptide, three oxidation reactions are involved; i) a hydroxylation at the benzylic position, ii) a hydroxylation at either the aromatic ring of Tyr or b-position of Ile, and iii) an oxidative cyclization. Considering the possible function of tyrosinase, UstQ may catalyze the oxidation of a phenol moiety, whereas the uncharacterized DUF3328 proteins UstYa and UstYb are most likely responsible for the remaining two-step oxidations. Notably, the UstY homolog was found to be the oxidation enzyme in the available biosynthetic gene cluster of fungal ribosomal pepteides such as asperipin-2a7 and phomopsin10, suggesting that the most intriguing macrocyclization of the core peptide is catalyzed by UstY homologs.

Mechanistic analysis of the modification enzymes for the aromatic side chain

1. Flavin dependent monooxygenases, UstF1 and UstF2

 We then turned our attention to the modification reactions to construct the amino acid-like side chain on the aromatic ring. On the basis of gene inactivation experiments, two Class B bifunctional flavin dependent monooxygenases,11UstF1 and UstF2, participate in the oxidative modification of 4. Oxidation of 4with a recombinant UstF1 and NADPH gave sulfoxide corresponding to 5. Subsequent incubation of the resultant 5 with UstF2 and NADPH gave two isomeric mixtures of oximes 7, of which structure was determined by NMR analyses. Treatment of 7 with 0.1% TFA readily afforded the corresponding geminal diol 9, a hydrate form of 8. Reduction of 9 with NaBH4 yielded 6, which was produced by the ustDdeletion mutant. To elucidate the catalytic mechanism of UstF2, we then conducted a time course analysis. At the beginning of the reaction, a monohydroxylated intermediate 10was produced. Taken together, the proposed reaction mechanism involves two rounds of oxidations followed by a decarboxylative elimination of the N-hydroxyl group (Figure 3). Similar oxime formation was reported in the caerulomycin A biosynthesis,12although a dehydration instead of a decarboxylative elimination occurs in the reaction.

2. Aminotransferase UstD

 UstD exhibits the similarity with an aminotransferase. Given the above speculation that a C3 nucleophile such as an enamine derived from alanine or aspartate likely reacts with a putative aldehyde intermediate 8 to give 1, UstD reaction with 9 was examined in the presence of PLP and aspartic acid. LC-MS analysis of the reaction product showed a new peak corresponding to 1. In the absence of 9, we detected a peak of an alanine derivative after derivatization by utilizing dansyl chloride (Figure 4). These results indicated that UstD catalyzed a b-decarboxylation of aspartic acid to yield an enamine followed by a C-C bond formation with aldehyde 8 to give 1 (Figure 4, path a). Although a simple b-decarboxylation was firmly established by an aspartate b-decarboxylase,13to our knowledge, condensation of the resultant enamine with an aldehyde in the same active site has not been reported.

 In this study, we applied heterologous expression and in vitro enzymatic assays to elucidate the biosynthetic pathway of ustiloxin B, which is the first identified fungal ribosomal peptides from filamentous fungi. The successful production of the cyclic peptide 2 in AO-ustAQYaYbshowed that heterologous expression was suitable to reconstitute the biosynthetic machinery of fungal ribosomal peptides. Most notably, the key oxidation enzyme UstY harboring an unknown DUF3328 motif were widely distribute in fungal genomes, showed their important roles for the biosynthesis. Additionally, in vitro characterization of tyrosine side chain modification enzymes revealed the novel chemical transformations. UstF1F2, two FAD dependent monooxygenases, catalyzed oxidation reactions on sulfur and nitrogen atom of aromatic side chain. UstD, a PLP dependent enzyme, catalyzed the decarboxylative C-C bond formation.

Acknowledgement

 We thank the Grant-in Aid for Scientific Research (A)15H01835 to H.O. for the support for this work. We thank Prof. Kazuo Shin-ya, late Dr. Miho Izumikawa, and Dr. Teppei Kawahara for giving us structural information of novel ustiloxins produced by gene knockout mutants.

References

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