Conspicuous degree of homology between the mitochondrial aspartate aminotransferases from chicken and pig heart.

The sequence of 40 amino acid residues at the amino terminus of mitochondrial aspartate aminotransferase from chicken heart differs in only 2 positions from the sequence of mitochondrial aminotransferase of pig heart. Close structural similarity had been suggested by previous data on syncatalytic sulfhydryl modifications (Gehring H., and Christen P. (1975) Biochem. Biophys. Res. Commun. $\text{63}$, 441–447). The cytosolic aspartate aminotransferases from the same two species have now been found to differ considerably in the mode of their syncatalytic modifications. The data suggest that the cytosolic and mitochondrial aspartate aminotransferases might have evolved at different organelle-specific rates.     

The amino acid sequence of cytosolic aspartate aminotransferase from human liver.

1. The cytosolic aspartate aminotransferase was purified from human liver. 2. The isoenzyme contains four cysteine residues, only one of which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. 3. The amino acid sequence of the isoenzyme is reported, as determined from peptides produced by digestion with trypsin and with CNBr, and from sub-digestion of some of these peptides with Staphylococcus aureus V8 proteinase. 4. The isoenzyme shares 48% identity of amino acid sequence with the mitochondrial form from human heart. 5. Comparisons of the amino acid sequences of all known mammalian cytosolic aspartate aminotransferases and of the same set of mitochondrial isoenzymes are reported. The results indicate that the cytosolic isoenzymes have evolved at about 1.3 times the rate of the mitochondrial forms. 6. The time elapsed since the cytosolic and mitochondrial isoenzymes diverged from a common ancestral protein is estimated to be 860 x 10(6) years. 7. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as a Supplementary Publication SUP 50158 (25 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.     

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     

Irreversible inactivation of aspartate aminotransferases during transamination with L-propargylglycine

L-Propargylglycine serves as an amino acid substrate in the transamination reaction catalyzed by both cytosolic and mitochondrial aspartate aminotransferases from pig heart. Incubation of these isoenzymes with L-propargylglycine alone did not result in the inactivation of these enzymes. However, the presence of 2-oxoglutarate or pyruvate caused gradual irreversible inactivation of these isoenzymes. The inactivation was greatly accelerated by the presence of formate ion. Inactivation of both isoenzymes with L-[2-¹⁴C]propargylglycine resulted in stoichiometric incorporation of the radioactive molecule. Drastic changes in the absorption and circular dichroic spectra of the enzymes which took place during the inactivation also indicated that the modification by L-propargylglycine is restricted to the active site of these isoenzymes.     

The covalent structure of mitochondrial aspartate aminotransferase from chicken. Identification of segments of the polypeptide chain invariant specifically in the mitochondrial isoenzyme

The primary structure of mitochondrial aspartate aminotransferase from chicken is reported. The enzyme is a dimer of identical subunits. Each subunit contains 401 amino acid residues; the calculated subunit molecular weight of the apoform is 44,866. The degree of sequence identity with the homologous cytosolic isoenzyme from chicken is 46%. A comparison of the primary structures of the mitochondrial and the cytosolic isoenzyme from pig and chicken shows that 40% of all residues are invariant. The degree of interspecies sequence identity both of the mitochondrial and the cytosolic isoenzyme from chicken and pig (86% and 83%, respectively) markedly exceeds that of the intraspecies identity between mitochondrial and cytosolic aspartate aminotransferase in chicken (46%) or in pig (48%). Based on these values, the duplication of the aspartate aminotransferase ancestral gene is estimated to have occurred approximately 1000 million years ago, i.e. at the time of the emergence of eukaryotic cells. By sequence comparison it is possible to identify amino acid residues and segments of the polypeptide chain that have been conserved specifically in the mitochondrial isoenzyme during phylogenetic evolution. These segments comprise about a third of the total polypeptide chain and appear to cluster in a certain surface region. The cluster carries an excess of positively charged residues which exceeds the overall charge difference between the cytosolic (pI approximately 6) and the mitochondrial isoenzyme (pI approximately 9).     

Distribution of aspartate aminotransferase activity in yeasts, and purification and characterization of mitochondrial and cytosolic isoenzymes from Rhodotorula minuta [corrected

The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.     

Interspecies comparison of cytosolic and mitochondrial aspartate aminotransferases. Evidence for a more conservative evolution of the mitochondrial isoenzyme.

The degree of structural similarity between the mitochondrial isoenzymes of aspartate aminotransferase from pig heart and chicken heart was determined by means of their immunological cross-reactivity and compared with the degree of similarity between the cytosolic isoenzymes from the same two species. Quantitative microcomplement fixation revealed a remarkable similarity of the two mitochondrial isoenzymes corresponding to an immunological distance of 104. The structures of the two cytosolic isoenzymes, on the other hand, diverge with an immunological distance of 203. The apparent conservatism of mitochondrial aspartate aminotransferase indicates additional evolutionary constraints on the structure of this organelle-confined isoenzyme.     

Structural and genetic relationships between cytosolic and mitochondrial isoenzymes

The most common type of genetic relationship between cytosolic and mitochondrial isoenzymes will probably be found to be divergent evolution from a common ancestral form. This is firmly established for the aspartate aminotransferases and less directly so in other cases. The two isoenzymes of aspartate aminotransferase have evolved at roughly equal rates at the level of total amino acid sequence but certain limited surface regions of the mitochondrial form have been much more highly conserved than corresponding regions in the cytosolic protein; these regions probably play a role in topogenesis of the mitochondrial isoenzyme. It is of interest that nearly all mitochondrial proteins are initially synthesised as precursors of molecular weight greater than the mature forms. In the case of aspartate aminotransferase, and possibly of other such isoenzymes, the N-terminus of the mature protein is nearly coincident with that of the cytosolic isoenzyme. Hence during evolution either the gene for the mitochondrial isoenzyme has gained an extra coding region for this N-terminal extension or, less likely, the structural gene for the cytosolic form has suffered a sizeable terminal deletion. Cytosolic and mitochondrial superoxide dismutases have not shared a common ancestral form as shown by the fact that their primary structures are completely unrelated. On the other hand, the mitochondrial and prokaryotic enzymes are clearly related. There is now, however, evidence to suggest that some prokaryotes possess a copper/zinc enzyme related to the eukaryotic cytosolic form. Hence the possibility arises that primitive prokaryotes possessed both proteins. The copper/zinc superoxide dismutase has been retained in the cytosol of eukaryotic cells and a few bacterial species.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. $\text{249}$, 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.     

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

Crystallization of pig mitochondrial aspartate aminotransferase by seeding with crystals of the chicken mitochondrial isoenzyme

Pig mitochondrial aspartate aminotransferase has been crystallized from polyethylene glycol solutions (M_r = 4000) with the aid of small seed crystals of the chicken mitochondrial isoenzyme. The “hanging drop” vapour diffusion technique was used. The unit cells of the pig and chicken mitochondrial isoenzymes are roughly isomorphous. Diffraction data have been collected to a resolution of 2.8 Å.     

Syncatalytic sulfhydryl group modification in mitochondrial aspartate aminotransferase from chicken and pig heart

One sulfhydryl group of the mitochondrial isoenzyme of aspartate aminotransferase from both chicken and pig heart exhibits syncatalytic reactivity changes similar to those found previously in the cytosolic isoenzyme from pig heart (Birchmeier, W., Wilson, K.J., and Christen, P. (1973) J. Biol. Chem. $\text{248}$, 1751–1759). The reactivity of the only titratable sulfhydryl group toward 5,5′-dithiobis-(2-nitrobenzoate) is at a minimum in the free pyridoxal and pyridoxamine form of the enzyme and is increased by approximately one order of magnitude when covalent enzyme-substrate intermediates are formed. The modification of the sulfhydryl group does not affect enzymatic activity. This finding supports the earlier conclusion that the syncatalytic reactivity changes are not due to a direct participation of this group in the active site but rather to conformational adaptations of the enzyme-coenzyme-substrate compound occurring in the catalytic mechanism of aspartate aminotransferases.     

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.     

The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.     

The active site of Sulfolobus solfataricus aspartate aminotransferase

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.     

Evolutionary relationships among aminotransferases. Tyrosine aminotransferase, histidinol-phosphate aminotransferase, and aspartate aminotransferase are homologous proteins

A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases. A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase. However, no significant similarity between these proteins and any of the other aminotransferases was detected. A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol-phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases. For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work-sheet. The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment. Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E. coli aspartate aminotransferase, rat and E. coli tyrosine aminotransferase, and S. cerevisiae and E. coli histidinol-phosphate aminotransferase are homologous proteins. Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5'-phosphate and the substrate, or appear to be essential for the conformation of the enzymes.     

Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes

Sulphydryl groups of mitochondrial aspartate aminotransferase from horse heart were titrated with 5,5'-dithiobis (2-nitrobenzoic acid). From analysis of peptic peptides, 378 amino acid residues (94.3% of the total) in the protein were identified. The results of amino acid sequence analysis are compared with those of cytosolic and mitochondrial aspartate aminotransferases from other sources.