CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI OF THE GENE CODING FOR MALATE-DEHYDROGENASE OF THE EXTREMELY HALOPHILIC ARCHAEBACTERIUM HALOARCULA-MARISMORTUI

The gene coding for the enzyme malate dehydrogenase (MDH) of the extre mely halophilic archaebacterium Haloarcula marismortui was isolated an d sequenced. The enzyme is composed of 303 amino acids, and its molecu lar mass is 32 638 Da. The deduced amino acid sequence of the enzyme w as found to be more similar to the sequence of L-lactate dehydrogenase (L-LDH) from various sources than to the sequence of other MDHs. The structural gene was cloned in the Escherichia coli expression vector p ET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction. Activation of the enzyme was obtaine d by increasing the salt concentration to 3 M NaCl. The recombinant pr otein was purified to homogeneity and shown to be indistinguishable fr om the native enzyme isolated from halobacteria. These findings presen t the first example of the successful expression of a halobacterial ge ne coding for a soluble protein in Escherichia coli and its recovery a s a functional enzyme. Site-directed mutagenesis was employed to modif y Arg100 on the enzyme to Gln. This modification produced an enzyme th at has considerably higher specificity for pyruvate (the substrate of L-LDH) than for oxaloacetate (the substrate of MDH). The mutation also caused a modification in the relative activities of the enzyme at dif ferent salt concentrations. The greater similarity of the amino acid s equence of the halobacterial MDH to that of L-LDHs than to that of MDH s sheds light on the molecular evolution of these enzymes.