2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. D-lactate dehydrogenases (n-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C-3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C-3 phenyl group. The 2-HADH from P. claussenii was found to have the highest k(cat)/K-m on PPA with 1,348.03 s(-1 )mM(-1) among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the n-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.