Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

Acylation of aspartate aminotransferase

1. Acetylation of aspartate aminotransferase from pig heart inhibits completely the enzymic activity when the coenzyme is in the amino form (pyridoxamine phosphate) or when the coenzyme has been removed, but not when the coenzyme is in the aldehyde form (pyridoxal phosphate). 2. The group the acylation of which is responsible for the inhibition has been identified with the in-amino group of a lysine residue at the coenzyme-binding site. Moreover, in the pyridoxamine-enzyme the amino group of the coenzyme is also acetylated. 3. The reactivity of the coenzyme-binding lysine residue is greatly different in the pyridoxamine-enzyme and in the apoenzyme, suggesting the possibility of an interaction of its in-amino group with pyridoxamine or with other groups on the protein.     

Reversible modification of amino groups in aspartate aminotransferase.

Amino groups in the pyridoxal phosphate, pyridoxamine phosphate, and apo forms of pig heart cytoplasmic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC .2.6.1.1) have been reversibly modified with 2,4-pentanedione. The rate of modification has been measured spectrophotometrically by observing the formation of the enamine produced and this rate has been compared with the rate of loss of catalytic activity for all three forms of the enzyme. Of the 21 amino groups per 46 500 molecular weight, approx. 16 can be modified in the pyridoxal phosphate form with less than a 50% change in the catalytic activity of the enzyme. A slow inactivation occurs which is probably due to reaction of 2,4-pentanedione with the enzyme-bound pyridoxal phosphate. The pyridoxamine phosphate enzyme is completely inactivated by reaction with 2,4-pentanedione. The inactivation of the pyridoxamine phosphate enzyme is not inhibited by substrate analogs. A single lysine residue in the apoenzyme reacts approx. 100 times faster with 2,4-pentanedione than do other amino groups. This lysine is believed to be lysine-258, which forms a Schiff base with pyridoxal phosphate in the holoenzyme.     

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.     

Mechanism of inactivation of Escherichia coli aspartate aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid

As a potential drug to treat neurological diseases, the mechanism-based inhibitor (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA) has been found to inhibit the γ-aminobutyric acid aminotransferase (GABA-AT) reaction. To circumvent the difficulties in structural studies of a S-ADFA-enzyme complex using GABA-AT, l-aspartate aminotransferase (l-AspAT) from Escherichia coli was used as a model PLP-dependent enzyme. Crystal structures of the E. coli aspartate aminotransferase with S-ADFA bound to the active site were obtained via cocrystallization at pH 7.5 and 8. The complex structures suggest that S-ADFA inhibits the transamination reaction by forming adducts with the catalytic lysine 246 via a covalent bond while producing 1 equiv of pyridoxamine 5'-phosphate (PMP). Based on the structures, formation of the K246-S-ADFA adducts requires a specific initial binding configuration of S-ADFA in the l-AspAT active site, as well as deprotonation of the ε-amino group of lysine 246 after the formation of the quinonoid and/or ketimine intermediate in the overall inactivation reaction.     

Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.     

Interaction of aspartate aminotransferase with amino acids

1. The capacity of various amino acids to convert the pyridoxal form of aspartate aminotransferase into the pyridoxamine form has been investigated. 2. Glutamate has the highest converting capacity; aspartate, α-aminopimelate, α-aminoadipate and other amino acids follow. 3. The converting capacity of the various amino acids assayed is connected with their structural features. 4. A possible role of amino acids as secondary substrates of aspartate aminotransferase is suggested.     

Three-dimensional structures of aspartate aminotransferase from Escherichia coli and its mutant enzyme at 2.5 A resolution

The structure of Escherichia coli aspartate aminotransferase complex with the inhibitor 2-methylaspartate, and that of the mutant enzyme in which an arginine was substituted for a lysine residue thereby forming a Schiff base with the coenzyme pyridoxal 5'-phosphate, were determined at 2.5 A resolution, by the molecular replacement method using the known structure of pig cytosolic aspartate aminotransferase. The enzyme catalyzes the reversible transamination between L-aspartate and alpha-ketoglutarate, and forms a dimeric structure of two identical subunits. Each subunit comprises two domains, a small and a large one. Although, in general, the overall and secondary structure of E. coli enzyme are similar to those of higher animals, some differences of enzymatic action between the enzyme from E. coli and those from higher animals could be explained on the basis of the X-ray structures and molecular mechanics calculation based on them.     

The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase

Reconstitution of wild-type apoaspartate aminotransferase from Escherichia coli with [4'-3H]pyridoxamine 5'-phosphate results in stereospecific release of the pro-S C-4' 3H to the solvent. The reaction follows first-order kinetics (t1/2 = 15 min at pH 7.5 and 25 degrees C), its rate constant being similar to that found previously with mitochondrial aspartate aminotransferase from chicken (Tobler, H.P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). Substituting the active site residue Lys258 by alanine via site-directed mutagenesis yields a catalytically inactive enzyme (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921). This mutant enzyme fails to release any measurable 3H from bound [4'-3H]pyridoxamine 5'-phosphate. The data are consistent with earlier proposals that Lys258 is indispensable for the ketimine/aldimine tautomerization, and corroborate the previous conclusion that 3H exchange from enzyme-bound pyridoxamine 5'-phosphate mechanistically corresponds to the deprotonation at C-4' of the ketimine intermediate during the transamination reaction.     

Characterization of pyridoxine auxotrophs of Escherichia coli: serine and pdxF mutants

At least six phenotypically distinct classes of mutants of Escherichia coli which require serine or pyridoxine or both can be isolated. Three of the six classes lack 3-phosphoserine-2-oxoglutarate aminotransferase. One of these classes contains WG5, a mutant previously characterized as containing the pdxF5 allele. The aminotransferase isolated from this mutant has been compared to that isolated from wild-type E. coli and found to have apparently normal affinity for pyridoxal 5'-phosphate, but reduced affinity for pyridoxamine 5'-phosphate.     

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α‐aminoadipate aminotransferase

α‐Aminoadipate aminotransferase (AAA‐AT) catalyzes the amination of 2‐oxoadipate to α‐aminoadipate in the fourth step of the α‐aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA‐AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2‐oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA‐AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine‐pyridoxal‐5‐phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate‐binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA‐AT/kynurenine aminotransferases.     

Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti.

A mutant of Rhizobium meliloti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.     

Site-directed mutagenesis of aspartate aminotransferase from E. coli

The gene for aspartate aminotransferase from E. coli (aspC) was subcloned into M13 phage and sequenced using the Sanger dideoxy method with synthetic oligonucleotide primers. A mutant gene was constructed using site-directed mutagenesis techniques in which the codon for the lysine that forms the Schiffs base with pyridoxal phosphate was replaced with one coding for alanine. The mutant gene was expressed under control of the Tac promoter to overproduce a mutant protein lacking enzymatic activity.     

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase. Activators and inhibitors

Measurement of the stereospecific release of the pro-S proton from C-4' of enzyme-bound pyridoxamine 5'-phosphate provides an experimental means to probe parts of the active site of aspartate aminotransferase independently of substrate turnover (Tobler, H. P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). The release of pro-S 3H from enzyme-bound [3H]pyridoxamine 5'-phosphate is 30,000 times faster than from free coenzyme. Enzyme-bound [3H]pyridoxine 5'-phosphate is not detritiated suggesting an essential role of the 4'-amino group. Formation of the unproductive complex of the [3H]pyridoxamine 5'-phosphate-enzyme with aspartate or glutamate results in a 400-fold acceleration of 3H release. In contrast, addition of borohydride or cyanoborohydride immediately stops 3H release. Experiments with a fluorescent reporter group and with differential chemical modifications indicate that the activating effect of aspartate on the release of 3H is accompanied by a shift of the so-called open/closed conformational equilibrium of the enzyme (Kirsch, J.F., Eichele, G., Ford, G. C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) toward the closed conformation; the inhibiting effect of borohydride and cyanoborohydride appears to be accompanied by a shift toward the open conformation. Apparently, at least part of the catalytic apparatus of aspartate aminotransferase becomes fully operative only in the closed conformation of the enzyme.     

Chemistry of the inactivation of 4-aminobutyrate aminotransferase by the antiepileptic drug vigabatrin

The chemical modification of pig liver 4-aminobutyrate aminotransferase by the antiepileptic drug 4-aminohex-5-enoate (Vigabatrin) has been studied. After inactivation by 14C-labeled Vigabatrin, the enzyme was digested with trypsin, and automated Edman degradation of the purified labeled peptide gave the sequence FWAHEHWGLDDPADVMTFSKK. Chymotryptic digestion of the tryptic peptide and sequencing of a resulting tripeptide identified the penultimate lysine residue of this peptide as the site of covalent modification. This lysine normally binds the coenzyme. Absorption spectroscopy demonstrated the absence of coenzyme from the tryptic peptide, and mass spectrometry showed its mass/charge ratio to be increased by 128. All of the bound coenzyme released after denaturation of the inactivated enzyme was as pyridoxamine phosphate. The structural nature of the modification is deduced, and mechanisms for its occurrence identified. Initially, 1 mol of radiolabeled inhibitor was bound per mol of monomer of the enzyme, although approximately half was released during denaturation and digestion, while the remainder was irreversibly bound. Coenzyme not released as pyridoxamine phosphate retained the absorbance characteristics of the aldimine, although the enzyme was completely inactive. Mass spectrometry of the sample of purified radiolabeled tryptic peptide revealed the presence of an approximately equal amount of a second fragment that contained no modification and from which the second lysine was absent, indicating that at the time of proteolysis the active site lysine was unaltered in 50% of the enzyme molecules.     

Crystalline enzyme.substrate complexes of asparate aminotransferase

Crystalline complexes of cytoplasmic aspartate aminotransferase of pig heart with the substrates L-glutamate and L-aspartate, and with other amino acids, have been prepared and polarized light absorption spectra have been measured. Striking differences in the directions of polarization of the absorption bands are seen. A complete half-transamination of pyridoxal phosphate to pyridoxamine phosphate by aspartate or by cysteine sulfinate can be demonstrated in the crystal as can the accumulation of a quinonoid intermediate with erythro-beta-hydroxyaspartate. X-ray diffraction studies show that the crystals with erythro-beta-hydroxyaspartate and alpha-methylaspartate are isomorphous with those of both alpha and beta subforms of the native enzyme.     

A heat-sensitive lysis mutant of Bacillus subtilis 168 with a low activity of pyruvate carboxylase.

A mutant of Bacillus subtilis which grew in complex medium at 30 degrees C but lysed at 45 degrees C has been isolated. It could only grow on minimal medium at 45 degrees C with added aspartate (20 microgram ml-1) but lysed if lysine (20 microgram ml-1) was also present. The requirement for aspartate was due to a low activity of pyruvate carboxylase; the site of the mutation (pyc) was linked (16% cotransducible using phage PBSI) to the pyrD locus, and the order of markers deduced was: pyrD-cysC-pyc. This defect appeared to lead to decreased synthesis of mesodiaminopimelic acid (mesoA2pm), an amino acid unique to peptidoglycan and its precursors. At the restrictive temperature the mutant accumulated uridine-5'-diphosphate N-acetylmuramyl-L-alanyl-D-glutamate, since meso A2pm is the next amino acid to be added to the growing peptide chain of peptidoglycan. This resulted in an inhibition of peptidoglycan synthesis, determined as a reduced incorporation of N-acetyl[14C]glucosamine. Peptidoglycan synthesis was not decreased if the mutant was grown in media containing aspartate but lacking lysine. The sensitivity to lysine may arise because (i) at 45 degrees C the mutant was starved for aspartate and hence mesoA2pm even when aspartate was present, since aspartate utilization, as estimated by the incorporation of [3H]aspartate into trichloroacetic acid precipitable material, was relatively inefficient; and (ii) this diminished level of mesoA2pm synthesis from aspartate was further curtailed since lysine inhibits one of the aspartokinases in B. subtilis. Thus, addition of lysine allowed protein synthesis and hence autolysin production to proceed whilst peptidoglycan synthesis remained inhibited. When autolysis was blocked, either indirectly by stopping protein synthesis through starvation of aspartate and lysine, or directly by introducing a lyt mutation, then shifting the mutant to 45 degrees C did not result in lysis but growth still ceased.     

Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease

A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.     

Identification of the Forms of Vitamin B6 Present in the Culture Media of “Vitamin B6 Control” Mutants

An Escherichia coli mutant resistant to isoniazid (WG497) contained 0.6 mumole of extracellular pyridoxamine and pyridoxamine phosphate in the early stationary phase. A suppressed lysine mutant (AT1024) contained 1.4 mumoles of pyridoxal phosphate under the same conditions. The internal concentration of vitamin B(6) was one-half of normal for AT1024 and increased fivefold for WG497.