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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
21999 43248 2008 6 PDF Available
Title
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
Abstract

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°C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50°C. 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.

Keywords
dye-linked d-lactate dehydrogenase; d-lactate; flavin adenine dinucleotide; Sulfolobus tokodaii; thermostable enzyme
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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
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 106, Issue 1, July 2008, Pages 16–21
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us