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3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

Lee, Philah, Raj, Subramanian Mohan, Zhou, Shengfang, Ashok, Somasundar, Edwardraja, Selvakumar, Park, Sunghoon
Biotechnology and bioprocess engineering 2014 v.19 no.1 pp. 1-7
EDTA (chelating agent), Escherichia coli, Pseudomonas denitrificans, beta-mercaptoethanol, catalytic activity, dithiothreitol, genes, iron, manganese, mercury, pH, silver, temperature
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 cₐₜ/K ₘ) 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 cₐₜ/K ₘ 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 cₐₜ/K ₘ values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn²⁺ increased the activity of 3HIBDHI significantly, whereas the presence of Fe²⁺, Hg²⁺ 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.