Trapping and Structural Elucidation of an Intermediate in the Macrophomate Synthase Reaction Pathway

Abstract

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.

Exploring the Biosynthesis of Natural Products and Their Inherent Suitability for the Rational Design of Desirable Compounds through Genetic Engineering

“…Recently, Hilvert and his co-workers elucidated the final reaction step, which progressed in a stepwise fashion involving a Diels-Alder reaction followed by decarboxylation, and concluding with a dehydration reaction, as illustrated in Scheme 1d. 35) The crystal structure of this protein visually revealed not only the locations of for substrate 1, pyruvate, and Mg 2þ , but also an interactive network system between the substrate, co-factor, and amino acid residues in the active site. 15) This model illustrates an overlapping molecular orbital between two substrates captured in the enzyme, which is indicative of a Diels-Alderase.…”

Biocatalytic Strategies towards [4+2] Cycloadditions

“…Another example of an intermolecular cyclase is the fungal enzyme macrophomate synthase . This enzyme catalyses a net‐cycloaddition between enolpyruvate ( 32 ) and 2‐pyrone 33 (Figure B).…”

“…This enzyme catalyses a net‐cycloaddition between enolpyruvate ( 32 ) and 2‐pyrone 33 (Figure B). However, the cycloaddition product 34 is not directly detectable as it undergoes immediate decarboxylation to the known intermediate 35 , which then dehydrates non‐enzymatically to reveal macrophomate . There are multiple indications that the initial enzyme‐catalysed cyclisation occurs in a stepwise manner rather than via a concerted Diels‐Alder mechanism.…”

The Diels–Alderase Never Ending Story

“…The catalytic mechanism of the MPS reaction was investigated extensively, showing that the reaction involves three separate steps: (i) decarboxylation; (ii) two carbon-carbon bond formations; and (iii) decarboxylation with concomitant dehydration [8,17]. Recently, it was found that the third step of the MPS reaction is decarboxylation to afford intermediate 10 and that the subsequent dehydration is not catalyzed by MPS [18]. Unstable intermediate 10 was released from active site and dehydrated to give macrophomate in a non-enzymatic manner.…”

“…Although intriguing observations that MPS itself can catalyze the aldol reaction have been reported (Scheme 21.3) [21], experimental evidence must be provided to evaluate the validity of calculation results. The recent finding that the first decarboxylation, the second C-C bond formation, and the third decarboxylation into 10 are rapid processes [18] suggested that it is difficult to distinguish a concerted from a stepwise process experimentally. 21 To support the involvement of a Diels-Alderase in this reaction, enzymatic conversion of 14 was examined.…”