In:
Protein Science, Wiley, Vol. 5, No. 5 ( 1996-05), p. 862-873
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 °C, i.e., ca. 60° below the optimal growth temperature of Thermotoga . Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 °C show satisfactory diffraction suitable for X‐ray analysis up to a resolution of 2.8 Å. 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 α‐helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas K M is unaltered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 °C; irreversible thermal denaturation becomes significant at ∼95 °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.
Type of Medium:
Online Resource
ISSN:
0961-8368
,
1469-896X
DOI:
10.1002/pro.5560050508
Language:
English
Publisher:
Wiley
Publication Date:
1996
detail.hit.zdb_id:
2000025-X
SSG:
12
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