Addition of racemase activity

As mentioned above, changing Cys188 to a weak proton donor, Ser, and introducing Cys instead of Gly74 brought about the inversion of enantioselectivity of

Table 3

Enantioselectivities of wild-type and mutant enzymes ?Ha AMDase

Table 3

Enantioselectivities of wild-type and mutant enzymes ?Ha AMDase

Enzyme

Ar

Yield (%)

Cys188Ser

Gly74Cys

Gly74Cys/Cys188Ser

> 99 17 37 60

99 (S) 50 (R) 0 (-) 94 (R)

Wild type

Cys188Ser

Gly74Cys

Gly74Cys/Cys188Ser

(XX

96 6 13 17

97 (R) 70 (S) 6 (R) 96 (S)

the enzyme. Then, changing the Gly74 to Cys keeping Cys188 as it stands, the active site of AMDase becomes the same as that of glutamate racemase. Thus, it is expected to have racemase activity to aryl propionate. The single mutant gene was prepared using the native gene as the template and appropriate primers. The mutant enzyme was produced by E. coli and purified in the same way as the native enzyme.

As expected, the Gly74Cys single mutant enzyme exhibited racemase activity to some arylpropionates as summarized in Table 4.30 In general, good substrates

Table 4

Racemase activity of Gly74Cys mutant AMdase

Table 4

Racemase activity of Gly74Cys mutant AMdase

Entry

Ar

R

Km

kcat

kcat /Km

Relative

(mM)

(s-1)

(s-1mM-1)

activity

1

Ph

ch3

17.4

1.34

0.077

100

2

2-Naphthyl

ch3

31.4

18.8

0.56

727

3

2-Thienyl

ch3

75.4

20.7

0.27

350

4

Ph

ch2ch3

16.3

0.30

0.018

24

5

Ph

CH2CH2CH3

-

-

-

0

6

Ph

CH(CH3)2

-

-

-

0

7

Ph

OH

55.6

0.59

0.011

14

8

Ph

NH2

3.90

0.018

0.0038

5

for decarboxylation are also good substrates for racemization. Interesting thing is that a-phenylbutyric acid was racemized by Gly74Cys mutant enzyme in spite of the fact that the corresponding malonic acid is totally inactive to decarboxylation (entry 4). It can be considered that the stereochemical repulsion might decrease in the case of the monobasic acid compared to the case of the dibasic acid.

Thus, decarboxylase of disubstituted malonic acid could be easily converted to racemase of the corresponding monobasic acid, in spite of the fact that decarboxylation and racemization are quite different from each other. The key for the success is the mechanistic consideration focusing on the fact that the intermediate of both reactions is the same type of enolate of monobasic carboxylic acid.

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