Detailed Reaction Mechanism of Macrophomate Synthase

Abstract

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.

Chemistry and Biology of Biosynthetic Diels–Alder Reactions

“…Thus, it is hardly surprising that a large portion of the experimental evidence for the Diels-Alder reaction in nature has been obtained for this class of compounds. In fact, both lovastatin and macrophomic acid, for which reported Diels-Alderase enzymes have been isolated, [7,8] are classified as polyketides.…”

“…[7,8] The isolation of these enzymes also 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 DielsAlder reaction in secondary metabolic biosynthetic pathways.…”

Total Synthesis and Biosynthesis of the Paraherquamides: An Intriguing Story of the Biological Diels–Alder Construction

“…It was interesting to observe that, in the case of cycloadducts 193 and 195, cleavage of the lactim ether with dilute HCl led to the production of the corresponding ring-opened amino esters (i.e., 197 from 193, Chart 39 (1,3) -type strain in compounds 193 and 195 caused by compression between the methyl group disposed on the b-face of the proline ring and the lactim ether methoxy group that is relieved upon ring-opening. In substrates 192 and 194, where the methyl group in the proline ring is on the a-face, the opportunity for A (1,3) -type strain is obviated by the anti-relationship between the lactim ether group and the methyl group.…”

“…1,2) There are just a few reports in the literature claiming the identification of an enzyme that catalyzes this most ubiquitous synthetic ring-forming reaction. [3][4][5][6] A possible explanation for this is that, enzymes generally catalyze reactions by stabilizing the structure and charge of the developing transition state. For most reactions subject to this stratagem of catalysis, both the starting substrate and the product differ significantly with respect to structure from the transition state; it is this fundamental difference that allows for turnover; i.e., both the product and the starting substrate must bind to the enzyme less tightly than the transition state structure.…”

Effective Use of Heterologous Hosts for Characterization of Biosynthetic Enzymes Allows Production of Natural Products and Promotes New Natural Product Discovery

“…[17][18][19][20] However, only five examples of biotransformation and their reaction mechanisms have been examined in detail using purified natural enzymes that catalyze a pericyclic reaction. Those five enzymes, SpnF in spinosyn biosynthesis, 21) solanapyrone synthase (Sol5), 22) LovB from lovastatin biosynthesis, 23,24) macrophomate synthase (MPS), 22,25) and riboflavin synthase (RibC) 26,27) were reported previously as enzymes that catalyze a Diels-Alder reaction based on their observed ability to promote a cycloaddition reaction (Chart 1). SpnF has been described as the first enzyme for which specific acceleration of a [4+2] cycloaddition reaction has been verified experimentally as its only observable function.…”