Cloning, site-specific mutagenesis, expression and characterization of full-length chloroplast NADP-malate dehydrogenase from Pisum sativum.
Chloroplast NADP-dependent malate dehydrogenase is regulated by a dithiol redox reaction. The assignment of the groups involved, requires the primary structure of the enzyme to be known. Using the polymerase chain reaction and the cDNA library of Pisum sativum, the sequence of the enzyme and its targeting signal was determined. The gene was cloned in Escherichia coli JM83 and expressed in E. coli JM83 and E. coli B at high yield. The determination of the physical properties of the gene product proves the recombinant protein to be indistinguishable from the enzyme purified from the plant. This holds true, in spite of the fact that the plant enzyme lacks 11 N-terminal residues. The lengths of the complete polypeptide chain of the recombinant enzyme and its transit peptide are 388 and 53 residues, respectively. The comparison of the sequences of the mature enzyme with those of known chloroplast NADP-MDH shows 83-95% identity, but with mitochondrial or bacterial MDH only approximately 20%. Reduction of the (inactive) oxidized enzyme with dithiothreitol allows mimicking of the in vivo activation. The reaction follows a consecutive second-order-kinetics mechanism. Guanidinium chloride (GdmCl) at concentrations below 0.4 M leads to a significant activation of the oxidized form of the enzyme. At [GdmCl] = 0.4-0.46 M, both oxidized and reduced NADP-MDH show highly cooperative changes in the hydrodynamic and spectral properties, indicating the synchronous breakdown of the quaternary, tertiary and secondary structures. Site-directed mutations C23A and C28A do not quench the regulatory properties of the enzyme; additional substitution of alanine for Cys206 and Cys376 renders the enzyme equally active in both the reduced and the oxidized state. Therefore, one can consider these residues, either alone or in combination with Cys23 and Cys28, as responsible for enzyme activation.
Reng
W
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Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Riessland
R
R
Scheibe
R
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Jaenicke
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D003460:Crystallization; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology*; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D009243:NAD; D004247:DNA; D008291:Malate Dehydrogenase