Reducing 3,4-dihydroxyphenylpyruvic acid to d-3,4-dihydroxyphenyllactic acid via a coenzyme nonspecific d-lactate dehydrogenase from Lactobacillus reuteri.

AIMS

The purpose of this work was to find an efficient enzyme to synthesize d-3,4-dihydroxyphenyllactic acid (d-DSS).

METHODS AND RESULTS

Nineteen lactic acid bacteria strains were screened for production of d-DSS using 3,4-dihydroxyphenylpyruvic acid (DPA) as a substrate. Lactobacillus reuteri JN516 exhibited the highest d-DSS yield. A nonspecific coenzyme, d-lactate dehydrogenase (d-LDH82319), from L. reuteri JN516 with high DPA reducing activity was identified. This enzyme reduced DPA to form d-DSS with excellent optical purity (enantioselectivity >99%). Its molecular weight was 35 kDa based on SDS-PAGE migration. The Michaelis-Menten constant (K ), turnover number (k ), and catalytic efficiency (k /K ) of d-LDH82319 for DPA were 0·09 mmol l , 2·17 s and 24·07 (mmol l )  s , respectively, with NADH as the coenzyme. The (K ), (k ) and (k /K ) of d-LDH82319 for DPA were 0·10 mmol l , 0·13 s and 1·30 (mmol l )  s , respectively, with NADPH as the coenzyme. The optimum temperature and pH of d-LDH82319 were 25°C and pH 8 respectively. Additionally, d-LDH82319 had a broad substrate range for alpha-keto acids, among which the activity of reducing pyruvate was the strongest; therefore, it belongs to the group of d-lactate dehydrogenases. d-LDH82319 and glucose dehydrogenase (GDH) were coexpressed to produce d-DSS from DPA.

CONCLUSIONS

d-LDH82319 from L. reuteri JN516 with high DPA reducing activity has the characteristics of a nonspecific coenzyme.

SIGNIFICANCE AND IMPACT OF THE STUDY

d-LDH82319 is the first reported coenzyme nonspecific d-lactate dehydrogenase with DPA-reducing activity. The coexpression system provided an effective method to produce d-DSS.