Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.
The isolation and sequence of a cDNA clone encoding the complete glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] of watermelon cotyledons are presented. Partial cDNA clones were synthesized in a three part strategy, taking advantage of the polymerase chain reaction technology with oligonucleotides based on directly determined amino acid sequences. Subsequently, the complete clone for gMDH was synthesized with a sense primer corresponding to the nucleotide sequence of the N-terminal end of pre-gMDH and an antisense primer corresponding to the adenylylation site found in the mRNA. The amino acids for substrate and cofactor binding identified by x-ray crystallography for pig heart cytoplasmic malate dehydrogenase are conserved in the 319-amino-acid-long mature plant enzyme. The pre-gMDH contains an N-terminal transit peptide of 37 residues. It has a net positive charge, lacks a long stretch of hydrophobic residues, and contains besides acidic amino acids a cluster of serine residues. This N-terminal extension is cleaved off upon association with or import into glyoxysomes. It contains a putative AHL topogenic signal for microbody import and has no sequence similarity to the 27-residue-long presequence of the watermelon mitochondrial malate dehydrogenase precursor.
Gietl
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Institute of Botany, Technical University of Munich, Federal Republic of Germany.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008291:Malate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D008291:Malate Dehydrogenase