title: Cloning, expression and characterization of 3-hydroxyisobutyrate dehydrogenase from Pseudomonas denitrificans ATCC 13867.
abstract: 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.
authors: Department of Chemical and Biomolecular Engineering, Pusan National University, Geumjeong-gu, Busan, Korea.
authors: Shengfang
authors: S
authors: Zhou
authors: Subramanian
authors: S
authors: Mohan Raj
authors: Somasundar
authors: S
authors: Ashok
authors: Selvakumar
authors: S
authors: Edwardraja
authors: Sun-Gu
authors: SG
authors: Lee
authors: Sunghoon
authors: S
authors: Park
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002614:Chelating Agents / Q000737:chemistry; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000737:chemistry; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D028561:Transition Elements / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D002614:Chelating Agents; D028561:Transition Elements; D019344:Lactic Acid; D004492:Edetic Acid; C031601:hydracrylic acid; D000429:Alcohol Oxidoreductases; C021398:3-hydroxyisobutyrate dehydrogenase
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