Enzymatic activity and partial purification of solanapyrone synthase: first enzyme catalyzing Diels–Alder reaction

Katayama, Kinya; Kobayashi, Tomonori; Oikawa, Hideaki; Honma, Mamoru; Ichihara, Akitami

doi:10.1016/s0167-4838(98)00040-5

Cited by 90 publications

(70 citation statements)

References 21 publications

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“…Although putative enzymes for unimolecular [4+2] cycloadditions have been reported (1)(2)(3)(4), no naturally occurring enzyme is known to catalyze a bimolecular Diels-Alder reaction (5,6). The generation of artificial Diels-Alderases has therefore been a longstanding and alluring goal for protein engineers.…”

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confidence: 99%

“…This approach was used, for example, to create DA_20_00, an enzyme that catalyzes the enantio-and diastereoselective Diels-Alder reaction between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate (1) and N,N-dimethylacrylamide (2) (21). The active site of a β-propeller scaffold was altered to bind the diene and dienophile for productive reaction and to stabilize the cycloaddition transition state electronically with appropriately placed hydrogen bond donors and acceptors (Fig.…”

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confidence: 99%

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Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

Preiswerk

Beck

Schulz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

119

101

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By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed DielsAlderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.biocatalysis | computational enzyme design | Diels-Alder reaction | laboratory evolution | enzyme mechanism

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confidence: 99%

mentioning

confidence: 99%

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

Preiswerk

Beck

Schulz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

119

101

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“…Also as described in the Introduction, solanapyrone A is a plant phytotoxin isolated together with other toxic metabolites from A. solani, the causal fungus of early potato blight (27,28). Solanapyrone A could inhibit the activities of mammalian pol ␤ and in the range of 50 -100 M. We should emphasize that solanapyrone A suppressed the activities of pol ␤ in vitro to the same extent as dideoxyTTP, a well known potent pol ␤ inhibitor (45).…”

Section: Discussionmentioning

confidence: 94%

“…Solanapyrone A, found as a novel inhibitor of pol ␤ and pol in this report, is a plant phytotoxin isolated together with other toxic metabolites from Alternaria solani, the causal fungus of early potato blight (27,28). The biochemical mode of action is still unknown, although there is a report on the detection of the enzymatic activity, isolation, and characterization of an enzyme catalyzing a Diels-Alder reaction (29).…”

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confidence: 82%

A Plant Phytotoxin, Solanapyrone A, Is an Inhibitor of DNA Polymerase β and λ

Mizushina¹,

Kamisuki²,

Kasai³

et al. 2002

Journal of Biological Chemistry

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Eukaryotic cells reportedly contain three replicative DNAdirected DNA polymerases (EC 2.7.7.7) (pol ␣, ␦, and ⑀), 1 mitochondrial DNA polymerase (pol ␥), and at least nine repair types of DNA polymerase (pol ␤, ␦, ⑀, , , , , , and ) (1-8), and we have screened for natural compounds that selectively inhibit each of these eukaryotic DNA polymerases and found several inhibitors (9 -15). The purpose was to use the compounds as tools and molecular probes to distinguish DNA polymerases and to clarify their biological and in vivo functions. In this study, we found an interesting fungus-produced compound that selectively inhibits the activities of pol ␤ and pol . Interestingly, the compound was found to be a plant phytotoxin, solanapyrone A, isolated from the causal fungus of early blight disease in potato. To our knowledge, such specific natural compounds have not been reported with the exception of the pol ␤-inhibitor, prunasin, which we reported previously (13). The compound was a stronger pol ␤-inhibitor than prunasin, and no pol -inhibitors have been reported.Pol ␤ is the lowest molecular mass (39 kDa) DNA polymerase lacking such intrinsic accessory activities as 3Ј-or 5Ј-exonuclease, endonuclease, dNMP turnover, or pyrophosphorolysis (16). Pol ␤ consists of an independently folded N-terminal 8-kDa domain and C-terminal 31-kDa domain (17,18). The N-terminal domain was originally characterized as a single-stranded DNA (ssDNA)-binding domain. It was then found to possess binding specificity for the 5Ј-phosphate in gapped DNA (19 -21) and a helix-hairpin-helix motif found in several other DNA repair enzymes (22,23). Recently, Matsumoto and Kim (24) demonstrated that pol ␤ catalyzes the removal of dRP from the AP endonuclease-incised AP site via ␤-elimination as opposed to hydrolysis, and that this dRP lyase activity resides in the 8-kDa domain of pol ␤. According to recent studies (7, 25), moreover, pol ␤ is known to have another family polymerase, pol . Pol ␤ has been present in all tissues examined and is generally expressed at a low level as are a number of other so-called constitutive "house-keeping" enzymes. Although the in vivo function of the family enzyme, pol , is unclear as yet, pol appears to work in a similar manner to pol ␤ (7). As to why a plant phytotoxin is a pol ␤-family-specific inhibitor, we presently are analyzing the structure and function of pol ␤ and pol using the inhibitor from two different view-points to understand the precise role of each of the polymerases in vivo and to develop a drug design strategy for cancer chemotherapy agents. We previously reported the three-dimensional structure of pol ␤ and the N-terminal 8-kDa domain (the DNA template-binding domain) with or without long chain fatty acids to further clarify the structure and function of pol ␤ (26). Because at least pol ␤ is an essential enzyme for nucleotide excision repair (1, 2), the plant phytotoxin may lead to blockage of the DNA repair systems of rescue cancer cells under clinical radiation-therapy or chemotherapy. The plan...

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“…1,2) Among of them, solanapyrones have been isolated as phytotoxic substances from the phytopathogenic fungus Alternaria solani. 3) We have reported the incorporation of the isotopically labeled biosynthetic precursors, prosolanapyrones I (1) and II (2), into (À)-solanapyrones, 4,5) and the partial purification and properties of the enzyme, solanapyrone synthase (SPS), 6) which is the first example of a natural Diels-Alderase. The biosynthetic pathway for solanapyrones is shown in Scheme 1.…”

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confidence: 99%

Purification and N-Terminal Amino Acid Sequence of Solanapyrone Synthase, A Natural Diels-Alderase fromAlternaria solani

Katayama

Kobayashi

Chijimatsu

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Enzymatic activity and partial purification of solanapyrone synthase: first enzyme catalyzing Diels–Alder reaction

Cited by 90 publications

References 21 publications

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

A Plant Phytotoxin, Solanapyrone A, Is an Inhibitor of DNA Polymerase β and λ

Purification and N-Terminal Amino Acid Sequence of Solanapyrone Synthase, A Natural Diels-Alderase fromAlternaria solani

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