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-m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K-m) of d-LDH82319 for DPA were 0 center dot 09 mmol l(-1), 2 center dot 17 s(-1) and 24 center dot 07 (mmol l(-1))(-1) s(-1), respectively, with NADH as the coenzyme. The (K-m), (k(cat)) and (k(cat)/K-m) of d-LDH82319 for DPA were 0 center dot 10 mmol l(-1), 0 center dot 13 s(-1) and 1 center dot 30 (mmol l(-1))(-1) s(-1), respectively, with NADPH as the coenzyme. The optimum temperature and pH of d-LDH82319 were 25 degrees 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.