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This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant ( k/ K) of 604.1 ± 71.1 mM/S on ( S)-3-hydroxyisobutyrate, but no detectable activity on ( R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD over NADP as a cofactor for its catalytic activity. The k/ K determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD at 37°C and pH 9.0. In addition to ( S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl- d, l-serine, and methyl-( S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k/ K values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn increased the activity of 3HIBDHI significantly, whereas the presence of Fe, Hg and Ag in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation. [ABSTRACT FROM AUTHOR]