Enzymatic activity catalysing exo-selective Diels–Alder reaction in solanapyrone biosynthesis

Oikawa, Hideaki; Katayama, Kinya; Suzuki, Yuichi; Ichihara, Akitami

doi:10.1039/c39950001321

Cited by 154 publications

(138 citation statements)

References 26 publications

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“…Literature that sets a precedent for the Diels-Alder reaction with 2-pyrone and an equivalent of pyruvate enolate (25) indicates that this route is not impractical. If the Diels-Alder route is operated, this could be the first purified enzyme catalyzing a Diels-Alder reaction (26,27). Although the simpler mechanism such as the Diels-Alder route explains well the multiple catalysis of macrophomate synthase, more data are necessary for establishing the mechanism of the second step.…”

Section: Discussionmentioning

confidence: 99%

Detailed Reaction Mechanism of Macrophomate Synthase

Watanabe¹,

Mie

Ichihara

et al. 2000

Journal of Biological Chemistry

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102

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Macrophomate synthase from the fungus Macrophoma commelinae IFO 9570 is a Mg(II)-dependent dimeric enzyme that catalyzes an extraordinary, complex five-step chemical transformation from 2-pyrone and oxalacetate to benzoate involving decarboxylation, C-C bond formation, and dehydration. The catalytic mechanism of the whole pathway was investigated in three separate chemical steps. In the first decarboxylation step, the enzyme loses oxalacetate decarboxylation activity upon incubation with EDTA. Activity is fully restored by addition of Mg(II) and is not restored with other divalent metal cations. The dissociation constant of 0.93 ؋ 10 ؊7 for Mg(II) and atomic absorption analysis established a 1:1 stoichiometric complex. Inhibition of pyruvate formation with 2-pyrone revealed that the actual product in the first step is a pyruvate enolate, which undergoes C-C bond formation in the presence of 2-pyrone. Incubation of substrate analogs provided aberrant adducts that were produced via C-C bond formation and rearrangement. This strongly indicates that the second step is two C-C bond formations, affording a bicyclic intermediate. Based on the stereospecificity, involvement of a Diels-Alder reaction at the second step is proposed. Incubation of the stereospecifically deuterium-labeled malate with 2-pyrones in the presence of malate dehydrogenase provided information for the stereochemical course of the reaction catalyzed by macrophomate synthase, indicating that the first decarboxylation provides pyruvate (Z)-[3-2 H]enolate and that dehydration at the final step occurs with anti-elimination accompanied by concomitant decarboxylation. Examination of kinetic parameters in the individual steps suggests that the third step is the rate-determining step of the overall transformation.

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Section: Discussionmentioning

confidence: 99%

Detailed Reaction Mechanism of Macrophomate Synthase

Watanabe¹,

Mie

Ichihara

et al. 2000

Journal of Biological Chemistry

Self Cite

102

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“…The ratio of the integration for the signals of deuterium at C17 to deuterium at C18 in the 2 Enzymatic activity was found in a cell-free extract of Alternaria solani, which catalyzed the conversion of 33 into solanapyrones A (26) and D (27) in 25 % yield with a ratio of the exo to endo cycloadduct of 53:47 . [21] A control experiment with denatured enzyme provided a 3:97 ratio of the exo to endo cycloadducts with only 10 % consumption of starting material. The observed stereoselectivity in the cell-free extract was interpreted as being indicative of enzymatic activity.…”

Section: Solanapyronesmentioning

confidence: 99%

Chemistry and Biology of Biosynthetic Diels–Alder Reactions

2003

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Nature's repertoire of biosynthetic transformations has recently been recognized to include the Diels-Alder cycloaddition reaction. Evidence now exists that there are enzymes that mediate the Diels-Alder reaction in secondary metabolic biosynthetic pathways. 2002 marked the 100th anniversary of Alder's birth and 75 years since the discovery of the Diels-Alder reaction. It would appear that living systems discovered and made use of this ubiquitously useful ring-forming reaction eons ago for the construction of complex natural products. In this Review an overview is given of all of the known classes of natural products (polyketides, isoprenoids, phenylpropanoids, alkaloids) that have been speculated to arise by a biological Diels-Alder reaction.

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“…This biological catalyst has high enantioselectivity and exo-selectivity, which cannot be achieved in chemical synthesis. 20 ) In conclusion, the P-450 inhibitor experiments is a powerful tool to reduce possible biosynthetic pathways that involve a number of oxidations. In the case that precursors are not found in the extracts, synthesis of advanced precursor is essential for collecting details of late biosynthetic pathways.…”

Section: Discussionmentioning

confidence: 99%

“…To estimate the optical purity of the products, HPLC analysis with continuous CD detection was undertaken. 20 ) On the basis of the UV and CD absorptions of enantiomerically pure natural solanapyrones, the optical purity of 8 was calculated as 92 ± 8°1o e.e. from the negative CD absorption at 300 nm.…”

Section: Diels-alderasementioning

confidence: 99%