Decarboxylation AMDase

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. ... 

Figure 10 Hammett plot of the of the AMDase-catalyzed decarboxylation of...

DNA sequence indicated that AMDase contains four cysteine residues located at 101, 148, 171 and 188 from amino terminal (Eig. 9). At least one of these four is estimated to play an essential role in the decarboxylation. The most effective way to determine which Cys is responsible to enzyme activity will be site-directed mutagenesis. To determine which amino acid should be introduced in place of active Cys, its role was estimated as illustrated in Eig. 13. One possibility is that... 

In the previous studies using inhibitors and additives, it became clear that AMDase requires no cofactors, such as biotin, coenzyme A and ATP. It is also suggested that at least one of four cysteine residues plays an essential role in asymmetric decarboxylation. One possibility is that the free SH group of a cysteine residue activates the substrate in place of coenzyme A. Aiming at an approach to the mechanism of the new reaction, an active site-directed inhibitor was screened and its mode of interaction was studied. Also, site-directed mutagenesis of the gene coding the enzyme was performed in order to determine which Cys is located in the active site. 

We screened for a potent inhibitor against the AMDase-catalyzed decarboxylation of a-methyl-a-phenylmalonic acid to give a-phenylpropionic acid. Among the compounds shown in Fig. 4 which have structures similar to the substrate. 

Fig. 5. Inhibition mode of a-bromophenylacetic acid against AMDase-catalyzed decarboxylation. Lineweaver-Burk plot in the presence of the acid A, 100 pM B, 20 pM C, 0 pM...

Fig. 7. Hammett plot of k at for the AMDase catalyzed decarboxylation of a series of X-phenyl-malonic acids...

Fig. 13. Proposed reaction mechanism of AMDase-catalyzed decarboxylation...

To clarify the characteristics of AMDase, the effects of additives were examined. The addition of ATP and coenzyme A (CoA) to the enzyme reaction mixture did not enhance the rate of decarboxylation. In the case of malonyl-CoA decarboxylase, ATP and substrate form a mixed anhydride, which in turn reacts with CoA to form a thiol ester of the substrate. In the case of AMDase, however, neither ATP nor CoA had any effect, so this mechanism is unlikely. It is well established that avidin is a potent inhibitor of biotin-enzyme complex formation [11,12]. In this case, addition of avidin had no influence on decarboxylase activity, indicating that AMDase is not a biotin-dependent decarboxylase. Thus, the cofactor requirements of AMDase are entirely different from known analogous enzymes, such as malonyl-CoA decarboxylases. 

TABLE 3.1 Reaction Specificity of AMDase and its Variant in Decarboxylation and Racemization... 

The reaction catalyzed by AMDase proceeds through enantioselective decarboxylation of the substrate and enantioface-selective protonation of the planar enolate intermediate by Cysl88 (Figure 3.10). 

In addition, we have succeeded in inversion of the enantioselectivity of AMDase using a rational design approach. Although the G74C/C188S mutant produced high yields of the (S)-enantiomer, the decarboxylation activity was much lower than wild-type AMDase. We have successfully improved the activity of an artificial (S)-selective AMDase variant using directed evolution. 

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]. 

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