Open AccessCloning, Expression and Characterization of 3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas denitrificans ATCC 13867
Author(s) -
Shengfang Zhou,
Subramanian Mohan Raj,
Somasundar Ashok,
Selvakumar Edwardraja,
SunGu Lee,
Sunghoon Park
Publication year - 2013
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0062666
Subject(s) - paracoccus denitrificans , escherichia coli , ethylenediaminetetraacetic acid , biochemistry , biology , nad+ kinase , enzyme , comamonas testosteroni , dehydrogenase , formate dehydrogenase , microbiology and biotechnology , chemistry , gene , cofactor , chelation , organic chemistry
The gene encoding an NAD + -dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to ( S )-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant ( K m ) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag + and Hg 2+ , completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.