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