Characterization of a novel dye-linked l-proline dehydrogenase from an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis

• DOI: 10.1007/s00253-010-2914-7
• Publication Date: 2010-10-13T23:32:33Z
• Authors: Takenori Satomura; Xiao-Dong Zhang; Yusuke Hara; Katsumi Doi; Haruhiko Sakuraba; Toshihisa Ohshima
• Keywords: 0301 basic medicine; Chromatography; Hot Temperature; Proline; Molecular Sequence Data; Coenzymes; Hydrogen-Ion Concentration; 3. Good health; Molecular Weight; Kinetics; 03 medical and health sciences; Enzyme Stability; Escherichia coli; Flavin-Adenine Dinucleotide; Proline Oxidase; Pyrobaculum; Amino Acid Sequence; Cloning, Molecular; Protein Multimerization; Sequence Alignment

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.