Inversion of enantioselectivity based on the reaction mechanism and homology

The reaction mechanism is becoming clear, i.e., enantiotopos-differentiating activation of pro-(R)-carboxyl group followed by decarboxylation to give the enolate form of the carboxylic acid and asymmetric protonation from Cys188 will give the final product. If this rather simple mechanism is actually working, then the inversion of the enantioselectivity of the reaction might be possible by changing the binding mode of the substrate or by shifting the key cysteine residue from the .«'-face to the re-face of the enolate intermediate. The former strategy seems rather difficult because the binding pocket of the aromatic ring and methyl group should be changed. On the other hand, the latter strategy seems more practical, as replacing the Cys188 residue with an amino acid that has little or no proton-donating ability is not difficult. Then, the introduction of a new proton donor (Cys) at a suitable position would bring about the expected inversion of enantioselectivity. In this case, the only thing to be done is to predict the position where the new Cys residue should be introduced.

However, this is not so easy without the tertiary structure of the enzyme. The possible clues are the homology search with functionally resembling enzymes and computer simulation of the tert-structure of the enzyme. The characteristic features of AMDase are: (i) the reaction proceeds via an enolate-type transition state,9 (ii) the cysteine residue plays an essential role917 and (iii) the reaction involves an inversion of configuration on the a-carbon of the carboxyl group.18

Some isomerases were found to have about 30% homology via multiple alignments using the PSI-BLAST program. These were glutamate racemase from Lactobacillus fermenti,21 aspartate racemase from Streptococcus thermophilus,22 hydantoine racemase from Pseudomanas sp. strain NS67123 and maleate isomerase from Alcaligenes faecalis.24 The important feature that is consistent for all these enzymes is the presence of Cys188. On the other hand, while all the isomerases have another cysteine residue at around 74, AMDase has no corresponding cysteine residue around this region as shown in Fig. 14.

The reaction mechanism for glutamate racemase has been studied exten-sively.25-27 It has been proposed that the key for the racemization activity is that the two cysteine residues of the enzyme are located on both sides of the substrate bound to the active site. Thus, one cysteine residue abstracts the a-proton from the substrate, while the other delivers a proton from the opposite side of the intermediate enolate of the amino acid. In this way, the racemase catalyzes the racemization of glutamic acid via a so-called two-base mechanism (Fig. 15).

The tertiary structure of glutamate racemase has already been resolved and it has also been shown that a substrate analog glutamine binds between two cysteine residues.28 These data enabled us to predict that the new proton-donating amino acid residue should be introduced at position 74 instead of Gly for the inversion of enantioselectivity of the decarboxylation reaction.

Glu racemase Asp racemase Hydantoine racemase Maleic acid isomerase AMDase







Cys184 Cys73

o oh

oh ho ho

Cys184 Cys73

Cys184 Cys73

Cys184 Cys73

Cys184 Cys73

Figure 15: Two-base mechanism of glutamate racemase.

First, we examined the enantioselectivity of the Cys188Ser mutant. The location of the proton donor does not change in this case although the proton-donating ability of Ser is weaker than that of Cys. Thus, the reaction is supposed to proceed slowly to result in the product of the same configuration as is the case of the reaction by the wild-type enzyme. However, the reality was entirely different. a-Methyl-a-thienyl- and a-methyl-a-(^-naphthyl)malonate gave the corresponding monobasic acids with the configuration opposite to that given by the native enzyme. This fact suggests that there are some other proton donors on the opposite side of the enantiomeric face of the intermediate enolate, although their effect is far smaller compared with that of Cys188. In the case of the Cys188Ser mutant, as the proton-donating ability of serine is weaker than that of cysteine, the hidden effect of other proton donors might be reflected in the product. Then, higher enantiomeric excess will be attained if the proton-donating ability of the amino acid residue located on the opposite side is stronger. Thus, Cys residue is introduced instead of the Gly74 of the native enzyme.

The Gly74Cys mutant was prepared via PCR using the plasmid that contains the gene-coding native AMDase. Although the change in amino acid is drastic, the mutant still exhibited some activity. As expected, the products were nearly racemic, if not entirely, in the case of the two substrates mentioned above. These results demonstrate that this position is effective to give a proton to the intermediate of the reaction.

If the proton-donating ability of the amino acid at 188 is weaker, then the enantioselectivity of the reaction will be reversed compared to that of native enzyme. As shown in Table 3, the absolute configuration of the products by this mutant is opposite to those of the products obtained by the native enzyme and the ee of the products dramatically increased to 94 and 96%, respectively. This inversion of the enantioselectivity of the reaction supports the reaction mechanism that the Cys188 of the native enzyme is working as the proton donor to the intermediate enolate form of the product.29

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