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.

All square: A {ZnO}2 square resting state in autocatalysis is revealed by NMR spectroscopy and endorsed by DFT calculations (see picture). Its dynamic behavior and reversible binding of excess iPr2Zn are defined.

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Macrophomate synthase (MPS) catalyzes the reaction of oxaloacetate and 2-pyrones to give, over multiple steps, substituted benzoates. We detected a transient intermediate in the course of this transformation by monitoring the total time course either spectroscopically at 305 nm or by 1H NMR. This species was trapped by quenching the reaction with acetonitrile and cooling the sample to 270 K; its structure was determined to be an allylic cyclohexadienol by a complete 2D NMR spectroscopic analysis. It is formed according to Michaelis−Menten kinetics at a rate that is as fast or faster than the decarboxylation of oxaloacetate, the first step in the reaction sequence. Dehydration of this compound to give macrophomate, which limits the rate of the overall process, is not catalyzed by MPS. Although these results clarify the final stages of MPS catalysis, they shed no light on the early C−C bond-forming step(s). As a consequence, other methods will be needed to resolve the controversy as to whether this enzyme functions as a natural Diels−Alderase.

Although the macrophomate synthase active site is rich in potential functional groups, site-directed mutagenesis shows that only three residues are absolutely required for catalysis of oxaloacetate decarboxylation and trapping of the resulting enolate with a 2-pyrone; the other residues that line the binding pocket are surprisingly tolerant to substitution.