Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.6.1.1 (aspartate aminotransferase)
21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a crystal derivatized with mercury ions. The protein model has been refined to a crystallographic R -value of 17.5% (R -free 22.6%). Each enzyme subunit consists of two domains, a large domain (residues 50-329) containing a seven-stranded predominantly parallel beta-sheet, surrounded by alpha-helices, and a small domain comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic dyad in the crystals, form the homodimeric molecule, which contains two equal active sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit and the large domain of the second subunit. The cofactor is anchored to the enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113, located at the N terminus of an alpha-helix. The pyridine nitrogen forms a hydrogen bond to the side-chain of the invariant residue Asp245. Electron density corresponding to a metal ion, most likely Na(+), was found in a tight turn at the surface of the enzyme. Structure analysis reveals that diaminopelargonic acid synthase belongs to the family of vitamin B6-dependent aminotransferases with the same fold as originally observed in aspartate aminotransferase. A multiple structure alignment of enzymes in this family indicated that they form at least six different subclasses. Striking differences in the fold of the N-terminal part of the polypeptide chain are one of the hallmarks of these subclasses. Diaminopelargonic acid synthase is a member of the aminotransferase subclass III. From the structure of the non-productive complex of the holoenzyme with the substrate 7-keto-8-aminopelargonic acid the location of the active site and residues involved in substrate binding have been identified.
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PMID:Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5'-phosphate-dependent enzymes. 1045 93

The rate of polypeptide chain elongation is up to one order of magnitude faster in prokaryotic cells than in eukaryotes. Here we report that the rates of in vitro refolding of orthologous prokaryotic and eukaryotic proteins correlate with their differential rates of biosynthesis. The mitochondrial and cytosolic aspartate aminotransferases of chicken and aspartate aminotransferase of Escherichia coli show pairwise sequence identities of 41-48% and nearly identical three-dimensional structures. Nevertheless, the prokaryotic enzyme refolded 6 times faster (at 25 degrees C) than the eukaryotic isoenzymes after denaturation in 6 m guanidine hydrochloride. Prokaryotic malate dehydrogenase and lactate dehydrogenase also renatured faster than their orthologous eukaryotic counterparts, suggesting that evolutionary pressure has adapted the rate of folding to the rate of elongation of polypeptide chains.
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PMID:Comparison of folding rates of homologous prokaryotic and eukaryotic proteins. 1078 76

X-ray crystal structures of three forms of human mitochondrial branched-chain aminotransferase (BCAT) were solved by molecular-replacement methods, using Escherichia coli BCAT as the search model. The enzyme is a homodimer and the polypeptide chain of each monomer has two domains. The small domain is composed of residues 1--175 and the large domain is composed of residues 176--365. The active site is close to the dimer interface. The 4'-aldehyde of the PLP cofactor is covalently linked to the epsilon-amino group of the active-site lysine, Lys202, via a Schiff-base linkage in two of the structures. In the third structure, the enzyme is irreversibly inactivated by Tris. The overall fold of the dimer in human mitochondrial BCAT is similar to the structure of two bacterial enzymes, E. coli BCAT and D-amino acid aminotransferase (D-AAT). The residues lining the putative substrate-binding pocket of human BCAT and D-AAT are completely rearranged to allow catalysis with substrates of opposite stereochemistry. In the case of human mitochondrial branched-chain aminotransferase, a hydrogen-bond interaction between the guanidinium group of Arg143 in the first monomer with the side-chain hydroxyl of Tyr70 in the second monomer is important in the formation of the substrate-binding pocket.
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PMID:The structure of human mitochondrial branched-chain aminotransferase. 1126 79

The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector. The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from [alpha-32P]UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA. The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome. These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family.
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PMID:Identification of the amino acid residue involved in rabbit hemorrhagic disease virus VPg uridylylation. 1136 64

Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852-16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate.
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PMID:Binding to chaperones allows import of a purified mitochondrial precursor into mitochondria. 1198 13

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.
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PMID:Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity. 1263 55

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.
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PMID:The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase. 1452 84

While the wild-type morning glory (Ipomoea tricolor) displays bright-blue flowers and dark-brown seeds, its spontaneous mutant, Blue Star, carrying the mutable ivory seed-variegated (ivs-v) allele, exhibits pale-blue flowers with a few fine blue spots and ivory seeds with tiny dark-brown spots. The mutable allele is caused by an intragenic tandem duplication of 3.3 kbp within a gene for transcriptional activator containing a basic helix-loop-helix (bHLH) DNA-binding motif. Each of the tandem repeats is flanked by a 3-bp sequence AAT, indicating that the 3-bp microhomology is used to generate the tandem duplication. The transcripts in the pale-blue flower buds of the mutant contain an internal 583-bp tandem duplication that results in the production of a truncated polypeptide lacking the bHLH domain. The mRNA accumulation of most of the structural genes encoding enzymes for anthocyanin biosynthesis in the flower buds of the mutant was significantly reduced. The transcripts identical to the wild-type mRNAs for the transcriptional activator were present abundantly in blue spots of the variegated flowers, whereas the transcripts containing the 583-bp tandem duplication were predominant in the pale-blue background of the same flowers. The flower and seed variegations studied here are likely to be caused by somatic homologous recombination between an intragenic tandem duplication in the gene encoding a bHLH transcriptional activator for anthocyanin biosynthesis, whereas various flower variegations are reported to be caused by excision of DNA transposons inserted into pigmentation genes.
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PMID:An intragenic tandem duplication in a transcriptional regulatory gene for anthocyanin biosynthesis confers pale-colored flowers and seeds with fine spots in Ipomoea tricolor. 1514 84

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.
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PMID:Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism. 1662 2

Hsc70 binds acid-unfolded mitochondrial aspartate aminotransferase (mAAT), forming either soluble or insoluble complexes depending on the relative concentrations of the proteins. Using partial proteolysis of Hsc70-mAAT complexes in combination with MALDI-TOF mass spectrometry, we have identified several potential Hsc70-binding regions in the mAAT polypeptide. Only one mAAT peptide was found bound to Hsc70 in the insoluble complexes while nine peptides arising from eight sequence regions of mAAT were found associated with Hsc70 in the soluble complexes. Most of these binding sites map to secondary structure elements, particularly alpha-helix, that are partly exposed on the surface of the folded structure. These results suggest that these peptide regions must not only be exposed but still in a flexible extended conformation in the mAAT folding intermediates recognized by Hsc70. Thus, for mAAT the discrimination between native and non-native structures by Hsc70 may rely more on the level of structure of the binding sites than on their degree of exposure to the solvent in the native structure.
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PMID:Identification of Hsc70 binding sites in mitochondrial aspartate aminotransferase. 1663 Nov 6


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