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

Takenori Satomura, Xiao Dong Zhang, Yusuke Hara, Katsumi Doi, Haruhiko Sakuraba, Toshihisa Ohshima

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12 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)1075-1082
Number of pages8
JournalApplied Microbiology and Biotechnology
Volume89
Issue number4
DOIs
Publication statusPublished - Feb 1 2011

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

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