AAT1, a gene encoding a mitochondrial aspartate aminotransferase in Saccharomyces cerevisiae.

We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.     

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X₅-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.     

Recombinant Expression, Purification, and Characterization of Three Isoenzymes of Aspartate Aminotransferase fromArabidopsis thaliana

Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plantArabidopsis thaliana.cDNA sequences encoding three of these AAT isoenzymes,asp1(mitochondrial),asp2(cytosolic), andasp5(plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods. Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species. Analysis of the recombinant proteins on denaturing PAGE gels indicated subunitM_rs of between 44 and 45 K. Kinetic parameters (K_mandk_cat) obtained for all four substrates (aspartate, α-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species. Further characterization of the purified recombinant enzymes alongside native enzymes fromA. thalianaleaf tissue on AAT activity gels confirmed the identity ofasp1andasp2as the mitochondrial and cytosolic AAT genes but indicated thatasp5may encode an amyloplastic rather than the chloroplastic enzyme.     

Structural basis for differences in substrate specificity of aspartate aminotransferase isoenzymes

X-Ray structural data concerning the substrate binding site of cytosolic chicken aspartate aminotransferase (AspAT) are reported. The structure of the complex of AspAT with the substrate-like inhibitor maleate has been refined at 2.2 A resolution. The lengths of hydrogen bonds between a bound molecule of maleate and side chains of amino acid residues in the active site are presented as well as other interatomic distances in the substrate binding site. The data obtained for the cytosolic AspAT have been compared with those for the mitochondrial chicken AspAT. It has been inferred that differences in substrate specificity of the AspAT isoenzymes are determined by interactions involving amino acid residues which are situated in the immediate vicinity of the active site and influence ionization or orientation of functional groups interacting with substrate. An explanation is suggested for different rates of transamination of aromatic amino acids in the active sites of the cytosolic and mitochondrial isoenzymes.     

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.     

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

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 miliaceum, 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 mitochondrial 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 are 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.     

Genomic structure,expression and evolution of the alfalfa aspartate aminotransferase genes

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.     

Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana

In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, including Arabidopsis thaliana . To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis. In addition to the previously identified ASN1 gene, we identified a novel class of asparagine synthetase genes in Arabidopsis ( ASN2 and ASN3 ) by functional complementation of a yeast asparagine auxotroph. The proteins encoded by the ASN2/3 cDNAs contain a Pur-F type glutamine-binding triad suggesting that they, like ASN1 , encode glutamine-dependent asparagine synthetase isoenzymes. However, the ASN2/3 isoenyzmes form a novel dendritic group with monocot AS genes which is distinct from all other dicot AS genes including Arabidopsis ASN1 . In addition to these distinctions in sequence, the ASN1 and ASN2 genes are reciprocally regulated by light and metabolites. Time-course experiments reveal that light induces levels of ASN2 mRNA while it represses levels of ASN1 mRNA in a kinetically reciprocal fashion. Moreover, the levels of ASN2 and ASN1 mRNA are also reciprocally regulated by carbon and nitrogen metabolites. The distinct regulation of ASN1 and ASN2 genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.     

Chromosomal localization of human aspartate aminotransferase genes by in situ hybridization

Summary The localization of the human genes for cytosolic and mitochondrial aspartate aminotransferase (AspAT) has been determined by chromosomal in situ hybridization with specific human cDNA probes previously characterized in our laboratory. The cytosolic AspAT gene is localized on chromosome 10 at the interface of bands q241–q251. Mitochondrial AspAT is characterized by a multigene family located on chromosomes 12 (p131–p132), 16 (q21), and 1 (p32–p33 and q25–q31). Genomic DNA from ten blood donors was digested by ten restriction enzymes, and Southern blots were hybridized with the two specific probes. Restriction fragment length polymorphism was revealed in only one case for cytosolic AspAT, with PvuII, while no polymorphism for mitochondrial AspAT was found.     

Evolutionary analysis of aspartate aminotransferases

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family. Correspondence to: C.J. Marshall     

Multiple isoforms and an unusual cathodic isoform of creatine kinase from channel catfish (Ictalurus punctatus)

In vertebrates, the creatine kinase (CK) family consists of two cytosolic and two mitochondrial isoforms. The two cytosolic isoforms are the muscle type (M-CK) and the brain type (B-CK). Here we report multiple CK isoenzymes in the diploid channel catfish (Ictalurus punctatus) with one unusual cathodic isoform that was previously found only in pathological situations in human. The cathodic CK isoform existed only in the channel catfish stomach, ovary, and spleen, but not in any other species analyzed such as tilapia, smallmouth bass, chicken, or rat. Two genes encode the multiple forms of the channel catfish M-CK cDNAs. M-CK1 has three alleles, M-CK1.1, M-CK1.2, and M-CK1.3, while M-CK2 has just one allele as determined by analysis of 17 cDNA clones and by allele-specific PCR. M-CK1 encodes a protein of 381 amino acids and the M-CK2 cDNA encodes a protein of 380 amino acids. The two cDNAs shared an 86% identity and both have the nine diagnostic boxes for cytosolic CKs and thus are of cytosolic origin. The M-CK1 gene was isolated, sequenced, and characterized and its promoter should be useful for transgenic research for muscle-specific expression.     

Identification of multi-gene families encoding isopentenyl diphosphate isomerase in plants by heterologous complementation in Escherichia coli

Two cDNAs encoding isopentenyl diphosphate isomerase (IPI) in Adonis aestivalis, Arabidopsis thaliana, and Lactuca sativa, and single examples from Oryza sativa and Tagetes erecta were identified. An analysis of these and other ipi leads us to suggest a separate origin for green algal and plant genes and propose that a single gene encodes plastid and cytosolic IPI in plants.     

Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins

We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP). Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs. Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features. ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants. ROC2 and ROC5 show elevated expression in flowers. Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression upon wounding. The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns. In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns. ROC4 is not expressed in roots, and is strongly induced by light. Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes. These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure. Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades. Known plant CyPs fall into three clades, one including the majority of higher-plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chloroplast CyPs.     

Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo

We have shown that the individual members of the plant gene family for glutamine synthetase (GS) are differentially expressed in vivo, and each encode distinct GS polypeptides which are targeted to different subcellular compartments (chloroplast or cytosol). At the polypeptide level, chloroplast GS (GS2) and cytosolic GS (GS1 and GSn) are distinct and show an organ-specific distribution. We have characterized full length cDNA clones encoding chloroplast or cytosolic GS of pea. In vitro translation products encoded by three different GS cDNA clones, correspond to the mature GS2, GS1, and GSn polypeptides present in vivo. pGS185 encodes a precursor to the chloroplast GS2 polypeptide as shown by in vitro chloroplast uptake experiments. The pGS185 translation product is imported into the chloroplast stroma and processed to a polypeptide which corresponds in size and charge to that of mature chloroplast stromal GS2 (44 kDa). The 49 amino terminal amino acids encoded by pGS185 are designated as a chloroplast transit peptide by functionality in vitro, and amino acid homology to other transit peptides. The cytosolic forms of GS (GS1 and GSn) are encoded by highly homologous but distinct mRNAs. pGS299 encodes the cytosolic GS1 polypeptide (38 kDa), while pGS341 (Tingey, S. V., Walker, E. L., and Coruzzi, G. M. (1987) EMBO. J. 6, 1-9) encodes a cytosolic GSn polypeptide (37 kDa). The homologous nuclear genes for chloroplast and cytosolic GS show different patterns of expression in vivo. GS2 expression in leaves is modulated by light, at the level of steady state mRNA and protein, while the expression of cytosolic GS is unaffected by light. The light-induced expression of GS2 is due at least in part to a phytochrome mediated response. Nucleotide sequence analysis indicates that chloroplast and cytosolic GS have evolved from a common ancestor and suggest a molecular mechanism for chloroplast evolution.     

Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis

Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A(4)B(4) configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Hb Nancy and Hb Osler: two distinct genetic variants with identical clinical and hemoglobin phenotype

Three hemoglobin variants (Hb Nancy, Osler and Fort Gordon), carrying the same Tyr→Asp substitution at position β145(HC2), have been independently described in 1975 in patients with marked polycythemia. The first one was found in a French Caucasian family from Lorraine, and the two others in African Americans. Two unrelated individuals with Hb Osler have been recently reinvestigated at the DNA level and surprisingly, in their β gene, codon 145 was found to be AAT which encodes for asparagine and not for aspartic acid, the aspartate at the protein level resulting, thus, from a very efficient posttranslational event. We reinvestigated a patient from the family of Hb Nancy and found that codon 145 was GAT, encoding for aspartate. This demonstrates that Hb Nancy is geneticaly distinct from Hb Osler despite an almost identical phenotype.