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

Satomura, Takenori, Zhang, Xiao-Dong, Hara, Yusuke, Doi, Katsumi, Sakuraba, Haruhiko, Ohshima, Toshihisa
Applied microbiology and biotechnology 2011 v.89 no.4 pp. 1075-1082
Escherichia coli, amino acid sequences, amino acids, electrons, enzymes, high performance liquid chromatography, molecular cloning, molecular weight, multigene family, pH
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 Δ1-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.