Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C₄ plant

Abstract

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliuceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondria1 AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT arc synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P . miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.Aspartate aminotransferase (AspAT) is a pyridoxal-phosphate-dependent enzyme which is distributed widely among plants, animals and microorganisms. This enzyme exists as isozymes in higher organisms. In animal cells, two distinct isozymes of AspAT can be found; one in the cytosol (c) and the other in the mitochondria (m). These isozymes function in amino acid metabolism, in the tricarboxylic acid cycle and in the malate-aspartate shuttle. The nucleotide sequences of the cDNA for both isozymes are known for the chicken [l, 21, pig [3,4], rat [5,6], mouse [7] and human [8,9]. The similarities of the amino acid sequences between these isozymes are close to 45% for all species studied. Apparently, the isozymes have evolved from a common ancestral AspAT by gene duplication. Studies of their three-dimcnsional structures have revealed some functionally and structurally important residues [lo -131. These residues are conserved among many AspAT isozymes.The leaf tissues of NAD-malic-enzyme-type C4 plants also contain cAspAT and mAspAT. These AspAT isozymes are important participants in the photosynthetic carbon assimilation pathway, being essential components in the movement of photosynthetic intermediates between mesophyll and Correspondence to M. Taniguchi, Department of Agricultural Chemistry, School of Agriculturc, Nagoya University, Nagoya, Japan 464-01Ahhrei~iations. AspAT, aspartate aminotransferase; c, cytosolic; m, mitochondrial.Erizyn7e. Aspartate aminotransferase (EC 2.6.1.1). Note. The novel nucleotide sequence data published here have been submitted to the EMBL sequence data bank(s) and are available under accession number(s) X63428 for pmAATl, X63429 for pcAAT2 and X63430 for pmAAT3.bundle sheath cells [14, 151. Both isozymes exhibi...

Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis.

“…AspAT genes have been cloned from severa1 plant species, including alfalfa, Panicum, soybean, carrot, and lupin (Gantt et al, 1992;Taniguchi et al, 1992;Turano et al, 1992;Wadsworth et al, 1993). Although only one or two cDNAs have been isolated from each of these other plant species, five different AspAT cDNAs have been cloned from Arabidopsis (Sch!Atz and Coruzzi, 1995;Wilkie et al, 1995).…”

Purification and Characterization of Aspartate Aminotransferase Isoenzymes from Rice Bran

“…The amino acid sequences predicted from cDNAs for mitochondrial AAT showed that mitochondrial AAT(s) are synthesized as precursor proteins that have a 28-amino-acid presequence. 6) In spite of recent progress in studies of plant AA Ts, detailed comparative studies on enzymatic properties of the isoenzymes of plant AA T from a single plant source with homogeneously purified preparations have not been done because of difficulty in the purification of the isoenzymes of plant AAT in a large enough amount to characterize them.…”

Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase, AAT4

“…The deduced amino acid sequence of the protein encoded by the pSAT2 open reading frame and AAT amino acid sequences from several plant and animal sources were compared. The pSAT2 sequence shares the highest level of similarity with the two Panicurn mitochondrial AAT proteins [21], 81.5~o and 82.0 ~o identity, respectively and with the rat mitochondrial AAT protein [ 12], 56.1 ~o identity.…”

“…The deduced amino acid sequence of the pSAT2-encoded protein was aligned with the protein sequences of the soybean plastid AAT [25] and the Panicum mitochondrial AAT3 [21] (Fig. 3).…”