EXTREMELY THERMOSTABLE L(-LACTATE DEHYDROGENASE FROM THERMOTOGA-MARITIMA - CLONING, CHARACTERIZATION, AND CRYSTALLIZATION OF THE RECOMBINANT ENZYME IN ITS TETRAMERIC AND OCTAMERIC STATE())

L(+)-lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophi lic 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 am ounts sufficient for physical characterization, and to analyze the mol ecular basis of its intrinsic stability, the gene was cloned and expre ssed functionally in Escherichia coli. Growth of the cells and purific ation 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. Crysta ls of the enzyme obtained at 4 degrees C show satisfactory diffraction suitable for X-ray analysis up to a resolution of 2.8 Angstrom. As sh own by gel-permeation chromatography, chemical crosslinking, light sca ttering, 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 sp ectroscopic characteristics (UV absorption, fluorescence emission, nea r- and far-UV CD) of the two species are indistinguishable. The calcul ated alpha-helix content is 45%, in accordance with the result of homo logy modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas K-M is unaltered. The extreme intr insic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 degrees C; irreversible thermal denaturation becomes significant at similar to 95 degrees C. The anomalous resistan ce toward chemical denaturation using guanidinium chloride and urea co nfirms 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.