Extremely thermostable L(+)‐lactate dehydrogenase from thermotoga maritima: Cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state

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.

Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima

“…Protein concentrations were determined using an absorption coefficient at 280 nm of 0.55 cm2 . mg-' ; this value, which was originally determined for the tetramer (Ostendorp et al, 1996), is also valid for the octamer. Activity measurements made use of the reduction of pyruvate in 0.1 M imidazole/HCl, pH 6.0 (25"C), 1 mM EDTA, 5 mM cysteamine, 0.6 mM sodium pyruvate, 0.02 mM Fru(1,6)P2, and 0.27 mM NADH at 55 "C.…”

“…Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas the K,,, is unchanged. The extreme intrinsic stability of the protein is reflected by its unaltered denaturation temperatures up to the upper limit of growth of the bacterium; irreversible thermal inactivation does not become significant below 95 "C (Ostendorp et al, 1996). The following experiments focus on the stability of the two enzyme species and their mode of association.…”

“…Purification of the enzyme. The purification of the enzyme followed procedures published elsewhere (Ostendorp et al, 1996). Active fractions containing the tetrameric and octameric enzyme were separated by gel-permeation chromatography using a 120-ml Superdex 200 pg column (1.6 cmX60 cm) preequilibrated with 50 mM Tris/HCI, pH 7.7, in the presence of 5 mM 2-aminoethanethiol (cysteamine), 1 mM EDTA, and 0.1 M NaCl.…”

“…Because of its low expression level in 7: maritima, a detailed characterization of the enzyme was not possible until the gene was cloned and expressed in E. coli (Ostendorp et al, 1993). The purification of the enzyme yielded a tetrameric and an octameric LDH species, which are indistinguishable with respect to their spectral properties and K,, value (Ostendorp et al, 1996). Thus, the two forms of the enzyme may serve as a model to investigate the mode of tetramer association, and its effect on the intrinsic stability of the enzyme in its two forms.…”

Activity, Stability and Structural Studies of Lactate Dehydrogenases Adapted to Extreme Thermal Environments

“…Strikingly, the DrLDH profile was found to be very narrow and centered at pH 7, as was observed with allosteric LDHs. 24,25 A pH value of 7 was therefore chosen to explore the thermal dependence of activity of Cg-, Dr-and TtLDHs in equivalent conditions. The results are plotted in Fig.…”