The Putative Diels−Alderase Macrophomate Synthase is an Efficient Aldolase

Abstract

We find that the putative Diels-Alderase macrophomate synthase (MPS) catalyzes the addition of pyruvate enolate, generated by decarboxylation of oxaloacetate, to a variety of aldehydes. Alkyl, aryl, and heteroaryl aldehydes are accepted as substrates, providing gamma-hydroxy-alpha-ketoacids in 35-95% yield with modest levels of stereochemical control. These aldol products, which are difficult to synthesize by other methods, are formed with efficiency comparable to that of macrophomate. Our results thus provide evidence that a two-step Michael-aldol pathway is a plausible alternative to the postulated [4 + 2] cycloaddition in the MPS-catalyzed addition of pyruvate enolate to 2-pyrones. They are also relevant to understanding the divergent evolution of type II pyruvate aldolases.

Biocatalytic Promiscuity

“…[76] Recently a promiscuous aldolase activity in MPS was discovered. [77,78] The aldol addition resulting Scheme 5. A: Macrophomate synthase (MPS) normally catalyses a Diels-Alder-like reaction to form macrophomate from 2-pyrone and oxaloacetate.…”

“…The aldol product was favoured 20-fold over a dehydrated product. [77,78] from promiscuous activity differs mechanistically from the native reaction because the Michael addition step is excluded. Various γ-hydroxy-α-ketoacids could be formed through MPS-catalysed aldol additions between oxaloacetate and various aldehydes.…”

“…A reaction with benzaldehyde and oxaloacetate resulted in a 70 % yield and a 44 % ee (S). [77] In a second publication, Hilvert's group showed that different protected 3-deoxysugar derivatives could be prepared by aldol additions with use of a pyruvate enolate, to afford protected aldoses with three to six carbons. [78] The enzyme showed a remarkable substrate tolerance, accepting various protecting groups (ether, acetal, allyl, benzyl, silyl ether and ester).…”

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

“…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.…”

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

“…23) Macrophomate synthase has been studied extensively by this author and his colleagues as the first Diels-Alder-catalyzing enzyme. 28) However, recent studies have suggested that the tandem Michael-Aldol reaction is a more plausible reaction pathway for this transformation, 29,30) Other multifunctional enzymes, Sol5 and RibC, have been proposed to participate in hydroxyl oxidation 22) and hydride transfer, 27) respectively, in addition to the [4+2] cycloaddition reactions. However, the concertedness of how these enzymes catalyze the multiple reactions has yet to be elucidated.…”