NDLI: Extremely thermostable L(+)-lactate dehydrogenase from Thermotoga maritima: cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state.

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Year: 1996 Volume: 5 Issue: 4

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Year: 1996 Volume: 5 Issue: 1

Extremely thermostable L(+)-lactate dehydrogenase from Thermotoga maritima: cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Ostendorp, R. Auerbach, G. Jaenicke, R.
Description	Country affiliation: Germany Author Affiliation: Ostendorp R ( Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany.)
Abstract	L(+)-lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195-201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 degrees C, i.e., ca. 60 degrees below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 degrees C show satisfactory diffraction suitable for X-ray analysis up to a resolution of 2.8 A. As shown by gel-permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near- and far-UV CD) of the two species are indistinguishable. The calculated alpha-helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unalatered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 degrees C; irreversible thermal denaturation becomes significant at approximately 95 degrees C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	5
Volume Number	5
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	1996-05-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Bacterial Proteins Chemistry Isolation & Purification Gram-negative Anaerobic Bacteria Enzymology L-lactate Dehydrogenase Protein Conformation Amino Acid Sequence Genetics Catalysis Circular Dichroism Cloning, Molecular Crystallization Crystallography, X-ray Escherichia Coli Hot Temperature Models, Molecular Molecular Sequence Data Molecular Weight Protein Denaturation Recombinant Fusion Proteins Sequence Alignment Sequence Homology, Amino Acid Spectrum Analysis Comparative Study Research Support, Non-u.s. Gov't Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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