Arylmalonate decarboxylase

Malonic acid ester synthesis is a classic but still one of the most important C—C bond-forming reactions, because it is widely applicable to various types of compounds and the reaction can be performed under mild conditions without special care to remove the trace amount of water and oxygen contained in the solvent. This reaction is especially useful in the synthesis of carboxylic acids. One important class of carboxylic acids is arylpropionates because optically active ones are known to have anti-inflammatory activity and other interesting physiological 

Although there were some trials to prepare optically active carboxylic acids via asymmetric decarboxylation, the optical yields of the products were not high enough for practical use. Thus, it is strongly desirable to find an enzyme which catalyzes asymmetric decarboxylation of arylmethylmalonates to give optically pure arylpropionates. 

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Although the absolute configurations of the products are opposite to that of antiinflammatory active compounds, and the substrate specificity is rather restricted as to the steric bulkiness around the reaction center, the enzyme system of A. bronchisepticus was proved to have a unique reactivity. Thus, detailed studies on the isolated enzyme were expected to elucidate some new interesting mechanism of the new type of decarboxylation. Thus, the enzyme was purified. (The enzyme is now registered as EC 4.1.1.76.) The molecular mass was about 24kDa. The enzyme was named as arylmalonate decarboxylase (AMDase), as the rate of the decarboxylation of phenylmalonic acid was faster than that of the a-methyl derivative. ... 

To obtain a better understanding of the reaction mechanism, some compounds that are considered to he intermediates were subjected to the reaction. Various reaction courses can be considered as illustrated in Fig. 21. Path A a-Methyltropic acid is oxidized to a-phenyl-a-methylmalonic acid. Then, the malonate is converted to optically active a-phenylpropionate hy arylmalonate decarboxylase. In order to confirm this assumption, incubation of the malonic acid with Rhodococcus sp. was carried out. The result obtained was the total recovery of the substrate, indicating that no decarboxylase is present in this bacterium. Path B a-Methyltropic acid is converted to racemic a-phenylpropionic acid, which is deracemized to optically active propionic acid. To examine the possibility of this route, racemic a-phenylpropionic acid was subjected to the reaction to observe... 

Lind MES, Himo F (2014) Theoretical study of reaction mechanism and stereoselectivity of arylmalonate decarboxylase. Acs Catal 4(11) 4153 160... 

Decarboxylation and Racemizatlon of Unnatural Compounds using ArtiUcial Enzymes Derived from Arylmalonate Decarboxylase... 

R. Obata and M. Nakasako, "Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cysl88fer mutant in the liganded form". Biochemistry 49,1963-1969 (2010). 

Y. Terao, K. Miyamoto and H. Ohta, "Introduction of single mutation changes arylmalonate decarboxylase to racemase". Chemical Communications 3600-3602 (2006). 

R. Kourist, Y. Miyauchi, D. Uemura and K. Miyamoto, "Engineering the promiscuous racemase activity of an arylmalonate decarboxylase". Chemistry—A European Journal 17, 557-563 (2011). 

S. Yoshida, J. Enoki, R. Kourist and K. Miyamoto, "Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance". Bioscience, Biotechnology, and Biochemistry 79,1965-1971 (2015). 

The above results prompted us to study in detail the isolated enzyme and gene in order to elucidate the mechanism of this type of decarboxylation. The enzyme was purified from the bacterium grown in a medium as described before. The enzyme was purified to about 300-fold to 377 U/mg protein (15% yield). Sodium dodecyl sulfate-polyamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) analysis showed that this enzyme was monomeric, the molecular mass being around 24 kDa. The enzyme was named as arylmalonate decarboxylase (AMDase) [16]. 

Decarboxylation of malonic acid derivatives is a well studied process in the biosynthesis of biomolecules such as long-chain fatty acids and polyketides. A decarboxylase that exhibits enantioselectivity for substituted malonates would be useful for producing ophcally active carboxylic acids, hi fact, malonyl-CoA decarboxylase does catalyze an enantioselective decarboxylation (Figure 3.2) [5], but malonyl-CoA is an unsuitable precursor for optically active substances. Instead, we focused on the prochiral-activated compoimd arylmalonate, an intermediate of malonic ester synthesis, to develop a method for enantioselective decarboxylation. Malonates are stable at room temperature but readily decompose to arylacetate and CO2 at high temperatures. This suggests that the decarboxylation of arylmalonate may occur naturally if arylmalonate acts as a substrate for a decarboxylase. 

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