CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI OF THE GENE CODING FOR MALATE-DEHYDROGENASE OF THE EXTREMELY HALOPHILIC ARCHAEBACTERIUM HALOARCULA-MARISMORTUI
F. Cendrin et al., CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI OF THE GENE CODING FOR MALATE-DEHYDROGENASE OF THE EXTREMELY HALOPHILIC ARCHAEBACTERIUM HALOARCULA-MARISMORTUI, Biochemistry, 32(16), 1993, pp. 4308-4313
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.