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 extremely halophilic archaebacterium Haloarcula marismortui was isolated and sequenced. The enzyme is composed of 303 amino acids, and its molecular mass is 32,638 Da. The deduced amino acid sequence of the enzyme was 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 pET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction. Activation of the enzyme was obtained by increasing the salt concentration to 3 M NaCl. The recombinant protein was purified to homogeneity and shown to be indistinguishable from the native enzyme isolated from halobacteria. These findings present the first example of the successful expression of a halobacterial gene coding for a soluble protein in Escherichia coli and its recovery as a functional enzyme. Site-directed mutagenesis was employed to modify Arg100 on the enzyme to Gln. This modification produced an enzyme that 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 different salt concentrations. The greater similarity of the amino acid sequence of the halobacterial MDH to that of L-LDHs than to that of MDHs sheds light on the molecular evolution of these enzymes.
Cendrin
F
F
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Chroboczek
J
J
Zaccai
G
G
Eisenberg
H
H
Mevarech
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001105:Archaea / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004284:Dogfish; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase