| RRC ID |
47519
|
| Author |
Satomura T, Zhang XD, Hara Y, Doi K, Sakuraba H, Ohshima T.
|
| Title |
Characterization of a novel dye-linked L-proline dehydrogenase from an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis.
|
| Journal |
Appl Microbiol Biotechnol
|
| Abstract |
The activity of a dye-linked L-proline dehydrogenase (dye-L: -proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5-9.0 for 30 min at 50 °C. The enzyme catalyzed L-proline dehydrogenation to Δ(1)-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for L-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-L-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far.
|
| Volume |
89(4)
|
| Pages |
1075-82
|
| Published |
2011-2-1
|
| DOI |
10.1007/s00253-010-2914-7
|
| PMID |
20936278
|
| MeSH |
Amino Acid Sequence
Chromatography
Cloning, Molecular
Coenzymes / metabolism
Enzyme Stability
Escherichia coli / genetics
Flavin-Adenine Dinucleotide / metabolism
Hot Temperature
Hydrogen-Ion Concentration
Kinetics
Molecular Sequence Data
Molecular Weight
Proline / metabolism
Proline Oxidase / chemistry
Proline Oxidase / genetics*
Proline Oxidase / isolation & purification
Proline Oxidase / metabolism*
Protein Multimerization
Pyrobaculum / enzymology*
Sequence Alignment
|
| IF |
3.53
|
| Times Cited |
11
|
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| Resource |
| General Microbes |
JCM 11548 |
