Peroxisomal alanine:glyoxylate aminotransferase deficiency in primary hyperoxaluria type I

Activities of alanine:glyoxylate aminotransferase in the livers of two patients with primary hyperoxaluria type I were substantially lower than those found in five control human livers. Detailed subcellular fractionation of one of the hyperoxaluric livers, compared with a control liver, showed that there was a complete absence of peroxisomal alanine:glyoxylate aminotransferase. This enzyme deficiency explains most of the biochemical characteristics of the disease and means that primary hyperoxaluria type I should be added to the rather select list of peroxisomal disorders.     

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.     

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.     

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.     

Capillary electrophoresis assay of alanine:glyoxylate aminotransferase activity in rat liver

We measured hepatic alanine:glyoxylate aminotransferase (AGT) activity using capillary electrophoresis. After rat liver homogenate was incubated in the presence of substrates and pyridoxal 5'-phosphate, the pyruvate and glycine produced by AGT were measured. The AGT activity was 10.02+/-0.31 micro mol pyruvate/h/mg protein and 10.21+/-0.15 micro mol glycine/h/mg protein. This method is relatively simple and shows superior sensitivity, allowing the measurement of enzyme activity in 5 micro g of protein. Therefore, it appears to be suitable for laboratory use and may also have advantages for measuring AGT activity in liver biopsy specimens.     

Intraperoxisomal form of alanine:glyoxylate aminotransferase in the peroxisomes of bird kidney

Alanine:glyoxylate aminotransferase has been reported to be present as the apo form in the peroxisomes and as the holo form in the mitochondria in chicken kidney. In contrast, the enzyme was found to be present as the holo form both in the peroxisomes and in the mitochondria in pigeon kidney, suggesting that birds are classified into two groups on the basis of intraperoxisomal form of kidney alanine:glyoxylate aminotransferase. In the kidney, the pigeon peroxisomal holo enzyme did not cross-react immunologically with the chicken peroxisomal apo enzyme.     

Reactions of human liver peroxisomal alanine:glyoxylate aminotransferase with beta-chloro-L-alanine and L-cysteine: spectroscopic and kinetic analysis

In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-l-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with l-alanine. On the other hand, during the reaction of AGT with l-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of l-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of l-cysteine is proposed. l-cysteine partitions between these two reactions with a ratio of ~2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of l-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of l-alanine and l-serine, respectively. Conversely, l-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of l-alanine and l-serine, respectively. This appears to be consistent with the calculated interaction energies of the l-cysteine, l-alanine and l-serine docked at the active site of AGT.     

Expression of alanine:glyoxylate aminotransferase gene from Saccharomyces cerevisiae in Ashbya gossypii

Two plasmids containing an autonomously replicating sequence from Saccharomyces cerevisiae were constructed. Using these vectors, the AGX1 gene encoding alanine:glyoxylate aminotransferase (AGT) from S. cerevisiae, which converts glyoxylate into glycine but is not present in Ashbya gossypii, was expressed in A. gossypii. Geneticin-resistant transformants with the plasmid having the kanamycin resistance gene under the control of the translation elongation factor 1 α (TEF) promoter and terminator from A. gossypii were obtained with a transformation efficiency of approximately 10–20 transformants per microgram of plasmid DNA. The specific AGT activities of A. gossypii pYPKTPAT carrying the AGX1 gene in glucose- and rapeseed-oil-containing media were 40 and 160 mU mg⁻¹ of wet mycelial weight, respectively. The riboflavin concentrations of A. gossypii pYPKTPAT carrying AGX1 gene in glucose- and rapeseed-oil-containing media were 20 and 150 mg l⁻¹, respectively. In the presence of 50 mM glyoxylate, the riboflavin concentration and the specific riboflavin concentration of A. gossypii pYPKTPAT were 2- and 1.3-fold those of A. gossypii pYPKT without the AGX1 gene.     

Diet and the frequency of the alanine:glyoxylate aminotransferase Pro11Leu polymorphism in different human populations

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) contains a Pro11Leu polymorphism that decreases its catalytic activity by a factor of three and causes a small proportion to be mistargeted from its normal intracellular location in the peroxisomes to the mitochondria. These changes are predicted to have significant effects on the synthesis and excretion of the metabolic end-product oxalate and the deposition of insoluble calcium oxalate in the kidney and urinary tract. Based on the evolution of AGT targeting in mammals, we have previously hypothesised that this polymorphism would be advantageous for individuals who have a meat-rich diet, but disadvantageous for those who do not. If true, the frequency distribution of Pro11Leu in different extant human populations should have been shaped by their dietary history so that it should be more common in populations with predominantly meat-eating ancestral diets than it is in populations in which the ancestral diets were predominantly vegetarian. In the present study, we have determined frequency of Pro11Leu in 11 different human populations with divergent ancestral dietary lifestyles. We show that the Pro11Leu allelic frequency varies widely from 27.9% in the Saami, a population with a very meat-rich ancestral diet, to 2.3% in Chinese, who are likely to have had a more mixed ancestral diet. F_ST analysis shows that the differences in Pro11Leu frequency between some populations (particularly Saami vs Chinese) was very high when compared with neutral loci, suggesting that its frequency might have been shaped by dietary selection pressure.     

Crystal structure of alanine:glyoxylate aminotransferase and the relationship between genotype and enzymatic phenotype in primary hyperoxaluria type 1

A deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is responsible for the potentially lethal hereditary kidney stone disease primary hyperoxaluria type 1 (PH1). Many of the mutations in the gene encoding AGT are associated with specific enzymatic phenotypes such as accelerated proteolysis (Ser205Pro), intra-peroxisomal aggregation (Gly41Arg), inhibition of pyridoxal phosphate binding and loss of catalytic activity (Gly82Glu), and peroxisome-to-mitochondrion mistargeting (Gly170Arg). Several mutations, including that responsible for AGT mistargeting, co-segregate and interact synergistically with a Pro11Leu polymorphism found at high frequency in the normal population. In order to gain further insights into the mechanistic link between genotype and enzymatic phenotype in PH1, we have determined the crystal structure of normal human AGT complexed to the competitive inhibitor amino-oxyacetic acid to 2.5A. Analysis of this structure allows the effects of these mutations and polymorphism to be rationalised in terms of AGT tertiary and quaternary conformation, and in particular it provides a possible explanation for the Pro11Leu-Gly170Arg synergism that leads to AGT mistargeting.     

Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1

We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C.J., P.J. Cooper, P.J. Wise, and P.R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly----Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro----Leu and Ile----Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.     

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.     

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.     

ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half- life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.     

Response of hepatic alanine:glyoxylate aminotransferase 1 to hormone differs among mammalia

The subcellular distribution and substrate specificity of hepatic alanine:glyoxylate aminotransferase 1 have been reported to differ among mammalia. In the present study, the response of this enzyme to hormone (glucagon) was found to differ among mammalia.