A Novel Flavin Adenine Dinucleotide(FAD) Containing D-Lactate Dehydrogenase from the Thermoacidophilic Crenarchaeota Sulfolobus tokodaii Strain 7 : Purification, Characterization and Expression in Escherichia coli(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZY

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概要

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• Dye-linked D-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80℃ for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50℃. The preferred substrate for this enzyme was D-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme's prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked D-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to D-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked D-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing D-lactate dehydrogenase.

• 2008-07-25

著者

• SATOMURA Takenori

  Department of Materials Science, Yonago National College of Technology

• SAKURABA Haruhiko

  Department of Applied Biological Science, Faculty of Agriculture & Graduate School of Agriculture, K

• OHSHIMA Toshihisa

  Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu Universi

• Sakuraba Haruhiko

  Department Of Agricultural Chemistry University Of Osaka Prefecture

• Satomura Takenori

  Department Of Materials Science Yonago National College Of Technology

• Kawakami Ryushi

  Analytical Research Center for Experimental Sciences, Saga University

• Ohshima T

  Microbial Genetics Division Institute Of Genetic Resources Faculty Of Agriculture Kyushu University

• Kawakami Ryushi

  Analytical Research Center For Experimental Sciences Saga University

• Ohshima Toshihisa

  Microbial Genetics Division Institute Of Genetic Resources Faculty Of Agriculture Kyushu University

• Ohshima Toshihisa

  Microbial Genetic Division Institute Of Genetic Resources Faculty Of Agriculture Kyushu University

• Satomura Takenori

  Department Of Applied Chemistry And Biotechnology Graduate School Of Engineering University Of Fukui

• Ohshima Toshihisa

  Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University

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