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Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.

ABSTRACT: 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.

SUBMITTER: Gietl C

PROVIDER: S-EPMC54410 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.

Gietl C C

Proceedings of the National Academy of Sciences of the United States of America 19900801 15

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 seq ...[more]

PMID: 2377615

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Project description:Oxygenic photoautotrophic bacteria, cyanobacteria, have the tricarboxylic acid (TCA) cycle, and metabolite production using the cyanobacterial TCA cycle has been spotlighted recently. The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) has been used in various studies on the cyanobacterial TCA cycle. Malate oxidation in the TCA cycle is generally catalyzed by malate dehydrogenase (MDH). However, Synechocystis 6803 MDH (SyMDH) is less active than MDHs from other organisms. Additionally, SyMDH uses only NAD+ as a coenzyme, unlike other TCA cycle enzymes from Synechocystis 6803 that use NADP+. These results suggest that MDH rarely catalyzes malate oxidation in the cyanobacterial TCA cycle. Another enzyme catalyzing malate oxidation is malic enzyme (ME). We clarified which enzyme oxidizes malate that originates from the cyanobacterial TCA cycle using analyses focusing on ME and MDH. In contrast to SyMDH, Synechocystis 6803 ME (SyME) showed high activity when NADP+ was used as a coenzyme. Unlike the Synechocystis 6803 mutant lacking SyMDH, the mutant lacking SyME accumulated malate in the cells. ME was more highly preserved in the cyanobacterial genomes than MDH. These results indicate that ME mainly oxidizes malate that originates from the cyanobacterial TCA cycle (named the ME-dependent TCA cycle). The ME-dependent TCA cycle generates NADPH, not NADH. This is consistent with previous reports that NADPH is an electron carrier in the cyanobacterial respiratory chain. Our finding suggests the diversity of enzymes involved in the TCA cycle in the organisms, and analyses such as those performed in this study are necessary to determine the enzymes. IMPORTANCE Oxygenic photoautotrophic bacteria, namely, cyanobacteria, have the tricarboxylic acid (TCA) cycle. Recently, metabolite production using the cyanobacterial TCA cycle has been well studied. To enhance the production volume of metabolites, understanding the biochemical properties of the cyanobacterial TCA cycle is required. Generally, malate dehydrogenase oxidizes malate in the TCA cycle. However, cyanobacterial malate dehydrogenase shows low activity and does not use NADP+ as a coenzyme, unlike other cyanobacterial TCA cycle enzymes. Our analyses revealed that another malate oxidation enzyme, the malic enzyme, mainly oxidizes malate that originates from the cyanobacterial TCA cycle. These findings provide better insights into metabolite production using the cyanobacterial TCA cycle. Furthermore, our findings suggest that the enzymes related to the TCA cycle vary from organism to organism and emphasize the importance of analyses to identify the enzymes such as those performed in this study.

Dataset Information

Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.

Publications

Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.

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