| RRC ID |
35617
|
| 著者 |
Zhou S, Mohan Raj S, Ashok S, Edwardraja S, Lee SG, Park S.
|
| タイトル |
Cloning, expression and characterization of 3-hydroxyisobutyrate dehydrogenase from Pseudomonas denitrificans ATCC 13867.
|
| ジャーナル |
PLoS One
|
| 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.
|
| 巻・号 |
8(5)
|
| ページ |
e62666
|
| 公開日 |
2013-1-1
|
| DOI |
10.1371/journal.pone.0062666
|
| PII |
PONE-D-12-33376
|
| PMID |
23658760
|
| PMC |
PMC3642240
|
| MeSH |
Alcohol Oxidoreductases / chemistry
Alcohol Oxidoreductases / genetics
Alcohol Oxidoreductases / metabolism*
Amino Acid Sequence
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
Catalytic Domain
Chelating Agents / chemistry
Cloning, Molecular
Edetic Acid / chemistry
Enzyme Stability
Hydrogen-Ion Concentration
Kinetics
Lactic Acid / analogs & derivatives*
Lactic Acid / metabolism
Molecular Docking Simulation
Molecular Sequence Data
Protein Structure, Secondary
Pseudomonas / enzymology*
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Structural Homology, Protein
Transition Elements / chemistry
|
| IF |
2.74
|
| 引用数 |
9
|
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| 原核生物(大腸菌) |
|
