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Title
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Cloning, sequencing, and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui.
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Authors
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F.Cendrin,
J.Chroboczek,
G.Zaccai,
H.Eisenberg,
M.Mevarech.
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Ref.
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Biochemistry, 1993,
32,
4308-4313.
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PubMed id
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Abstract
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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.
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