Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.     

Immunological heterogeneity of hepatic alanine:glyoxylate aminotransferase in primary hyperoxaluria type 1

Immunoblotting of human liver sonicates, after SDS-polyacrylamide gel electrophoresis, demonstrated the presence of a 40 kDa protein, corresponding to the subunit of alanine:glyoxylate aminotransferase, in six controls and three patients with primary hyperoxaluria type 1 (peroxisomal alanine:glyoxylate aminotransferase deficiency). This immunoreactive 40 kDa protein was absent in a further nine patients. Subcellular fractionation of patients' livers showed that the 40 kDa protein, when present, was located mainly in the peroxisomes. In a heterozygote liver, the 40 kDa protein was also mainly peroxisomal and paralleled the distribution of alanine:glyoxylate aminotransferase activity.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate/serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal human liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

Developmental profiles and properties of hepatic peroxisomal apo- and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis

Alanine:glyoxylate aminotransferase was present as the apoenzyme in the peroxisomes and as the holoenzyme in the mitochondria in chick embryos. The peroxisomal enzyme predominated in the early stage and gradually decreased during embryonic development and disappeared after hatching. In contrast, the mitochondrial enzyme gradually increased and predominated in the later stage of chick embryos. Peroxisomal alanine:glyoxylate aminotransferase in chick embryos was a single peptide with a molecular weight of about 40,000. The enzyme differed from the mitochondrial enzyme in the embryos, and mammalian alanine:glyoxylate aminotransferases 1 (with a molecular weight of about 80,000 with two identical subunits) and 2 (with a molecular weight of about 200,000 with four identical subunits) in molecular weights and immunological properties. Mitochondrial alanine:glyoxylate aminotransferase in chick embryos had an identical molecular weight and immunologically cross-reacted with mammalian mitochondrial alanine:glyoxylate aminotransferase 2. Pyridoxal 5'-phosphate dissociated easily from the peroxisomal enzyme saturated with pyridoxal 5'-phosphate. Hepatic aspartate:2-oxoglutarate aminotransferase and alanine:2-oxoglutarate aminotransferase in chick embryos, and hepatic alanine:glyoxylate aminotransferases in different animal species were all present as the holoenzyme.     

Construction, purification and characterization of untagged human liver alanine-glyoxylate aminotransferase expressed in Escherichia coli

His-tagging is commonly used to aid and expedite the purification of recombinant proteins. It is commonly assumed, though less frequently tested, that the His-tag affects neither the structure nor the stability of the protein. Alanine:glyoxylate aminotransferase (AGT) is a peroxisomal pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the transamination of alanine and glyoxylate to pyruvate and glycine. AGT is a clinically relevant enzyme whose deficiency causes an inherited rare metabolic disorder named primary hyperoxaluria type I. Until now, the structure and function of this enzyme have been studied using recombinant wild-type AGT and variants purified using a hexa-histidine tag. However, the study of the functional roles of the N- and C-termini in the dimerization process and on the import into the peroxisome, respectively, requires the preparation of human liver AGT without histidine tags. We report for the first time the expression of untagged AGT together with a new rapid protocol for its purification. In addition, the kinetic parameters for the forward and reverse transamination catalyzed by untagged AGT as well as the spectroscopic features, the K(D(PLP)), the pH and thermal stability of the enzyme in the holo- and apo-form have been determined. This investigation will be the starting point for a detailed understanding of the contributions of the N- and C-termini on the dimerization and folding of AGT, and on its import into the peroxisome. This is prerequisite to understand how pathological mutations affect the proper native quaternary and tertiary structure, stability, and targeting of the enzyme.     

Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications

Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5'-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the K(eq),(overall) of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5'-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT-PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (approximately 0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT-PMP into AGT-PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.     

Serine: glyoxylate, alanine:glyoxylate, and glutamate:glyoxylate aminotransferase reactions in peroxisomes from spinach leaves

Two different aminotransferases, that have glyoxylate as the amino acceptor, have specific activities of 1 to 2 mumol . min-1 . mg of protein-1 in the isolated peroxisomal fraction from spinach leaves. Their properties were evaluated after separation on a hydroxylapatite column. Both enzymes had a Km for glyoxylate of 0.15 mM and an amino acid Km of 2 to 3 mM. Reactions proceeded by a Ping Pong Bi Bi mechanism. Serine:glyoxylate aminotransferase was relatively specific for both substrates and could only be slightly reversed with 100 mM glycine, although the Ki of glycine was 33 mM. The glutamate:glyoxylate amino-transferase protein was equally active in catalyzing an alanine:glyoxylate aminotransferase reaction, but the reverse reactions with 100 mM glycine were hardly measureable, although the Ki (glycine) was 8.7 mM. Protection against hydroxylamine inhibition from reaction with pyridoxal phosphate was used to investigate the specificity of amino acid binding. Substrate amino acids protected at about the same concentration as their Km, while glycine protected at its Ki concentration. Thus, the nearly irreversible catalysis with glycine is not due to a failure to bind glycine. The significance of a peroxisomal alanine:glyoxylate aminotransferase activity has not been incorporated into schemes for the oxidative photosynthetic carbon cycle.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.     

The evolution of peroxisomal and mitochondrial alanine: glyoxylate aminotransferase 1 in mammalian liver

Immunological distances of alanine: glyoxylate aminotransferase 1 (serine:pyruvate aminotransferase) in mitochondria or peroxisomes from eight different mammalian liver were determined with rabbit anti-serum against the mitochondrial enzyme of rat liver by microcomplement fixation. Results suggest that heterotopic alanine:glyoxylate aminotransferase 1 are orthologous proteins and their subcellular localization and substrate specificity changed during rapid molecular evolution.     

Alanine: glyoxylate aminotransferase 1 is present in the peroxisomes of guinea pig kidney

The subcellular distribution of alanine: glyoxylate aminotransferase 1 in guinea pig and rabbit kidneys was examined by centrifugation in a sucrose density gradient. The enzyme was located in the peroxisomes of guinea pig kidney and cross-reactive with the antibody against rat liver alanine: glyoxylate aminotransferase 1. This is the first report on the presence of the enzyme in the peroxisomes of mammalian kidney. The enzyme was found to be located in the mitochondria but not in the peroxisomes in rabbit kidney.     

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.     

Alanine:glyoxylate aminotransferase is present as the apoenzyme in the peroxisomes of chicken kidney

The subcellular distribution of alanine:glyoxylate aminotransferase in chicken kidney was examined by centrifugation in a sucrose density gradient. The enzyme was found to be present as the apoform in the peroxisomes and as the holoform in the mitochondria. Alanine:glyoxylate aminotransferase in different mammalian kidneys were all present as the holoenzyme in the mitochondrial and soluble fractions.     

Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′‐phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP‐binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT‐pyridoxamine 5′‐phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300‐ to 500‐fold decrease in both the rate constant of L‐alanine half‐transamination and the k_cat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc.     

Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase

Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects. Gly161 is a highly conserved residue whose mutation to Arg, Cys or Ser is associated with PH1. Here we investigated the molecular bases of the AGT deficit caused by Gly161 mutations with expression studies in a mammalian cellular system paired with biochemical analyses on the purified recombinant proteins. Our results show that the mutations of Gly161 (i) strongly reduce the expression levels and the intracellular half-life of AGT, and (ii) make the protein in the apo-form prone to an electrostatically-driven aggregation in the cell cytosol. The coenzyme PLP, by shifting the equilibrium from the apo- to the holo-form, is able to reduce the aggregation propensity of the variants, thus partly decreasing the effect of the mutations. Altogether, these results shed light on the mechanistic details underlying the pathogenicity of Gly161 variants, thus expanding our knowledge of the enzymatic phenotypes leading to AGT deficiency.     

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

Identification of mammalian aminotransferases utilizing glyoxylate or pyruvate as amino acceptor. Peroxisomal and mitochondrial asparagine aminotransferase

The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity.