Enhancing (L)-isoleucine production by thrABC overexpression combined with alaT deletion in Corynebacterium glutamicum.

L-isoleucine is synthesized from 2-ketobutyrate and pyruvate in Corynebacterium glutamicum, and the supplies of these two precursors are important for L-isoleucine synthesis. C. glutamicum YILWΔalaT with alaT gene deletion (encoding alanine aminotransferase, a principal enzyme for L-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrABC genes from Escherichia coli (encoding bifunctional aspartate kinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILWΔalaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILWΔalaT, and YILWΔalaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher L-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILWΔalaT excreted lower concentrations of L-lysine, L-alanine, and L-valine. YILWΔalaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to L-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly.

[Primary hyperoxaluria]. / Hyperoxalurie primitive.

Primary hyperoxalurias are rare recessive inherited inborn errors of glyoxylate metabolism. They are responsible for progressive renal involvement, which further lead to systemic oxalate deposition, which can even occur in infants. Primary hyperoxaluria type 1 is the most common form in Europe and is due to alanine-glyoxylate aminostransferase deficiency, a hepatic peroxisomal pyridoxin-dependent enzyme. Therefore primary hyperoxaluria type 1 is responsible for hyperoxaluria leading to aggressive stone formation and nephrocalcinosis. As glomerular filtration rate decreases, systemic oxalate storage occurs throughout all the body, and mainly in the skeleton. The diagnosis is first based on urine oxalate measurement, then on genotyping, which may also allow prenatal diagnosis to be proposed. Conservative measures - including hydration, crystallization inhibitors and pyridoxine - are safe and may allow long lasting renal survival, provided it is given as soon as the diagnosis has been even suspected. No dialysis procedure can remove enough oxalate to compensate oxalate overproduction from the sick liver, therefore a combined liver and kidney transplantation should be planned before advanced renal disease has occurred, in order to limit/avoid systemic oxalate deposition. In the future, primary hyperoxaluria type 1 may benefit from hepatocyte transplantation, chaperone molecules, etc.

Case report: Alanine aminotransferase deficiency detected in a patient with chronic hepatitis C.

We report a case of alanine aminotransferase (ALT) deficiency in a 68-year-old Japanese female with chronic hepatitis C. The serum was positive for antibody to hepatitis C virus (HCV) and HCV-RNA. Liver biopsy showed histological evidence of chronic active hepatitis. The level of serum aspartate aminotransferase (sAST) was elevated, but sALT was extremely low. The patient was followed up for her serum aminotransferase levels for 1.5 years under the treatment with ursodeoxycholic acid. The low sALT level persisted during all the follow-up period. The ALT activity in liver tissue was also decreased. Based on these findings, ALT deficiency was suspected. sALT activity was also found to be low in her two sons. This latter finding suggests the hereditary character of this abnormality.

Potential for bilateral nephrectomy to reduce oxalate release after combined liver and kidney transplantation for primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH-1) is frequently associated with end stage renal failure due to urinary calculi, obstructive uropathy and interstitial deposits of calcium oxalate. The currently accepted treatment for PH-1 is liver transplantation to replace the deficient enzyme peroxisomal alanine glycoxylate aminotransferase (AGT) and a simultaneous renal transplant to restore renal function. The transplanted kidney may become significantly impaired or fail when systemic calcium oxalate is eliminated by renal excretion. The native kidneys are a major source of this oxalate. This study was undertaken to determine whether there is a difference in oxalate clearance following combined liver-kidney transplant in patients with PH-1 by comparing the effect of native kidney nephrectomy at the time of transplantation against leaving the native kidneys in situ. Regression analysis was used to compare daily urinary oxalate excretion corrected for body surface area. There was a significant reduction in urinary oxalate excretion (P < 0.05) in the patient who had undergone bilateral nephrectomy compared to the patient whose native kidneys remained in situ for the first 100 d following combined liver and kidney transplantation. No difference was observed in the serum oxalate levels between patients over the same period or in the renal function assessed by creatinine clearance corrected for body surface area. Total body oxalate load was not determined in this study. A larger study should be undertaken to examine the benefits of nephrectomy in reducing oxalate deposition in recently inserted allografts for patients with PH-1.

Molecular characterization and clinical use of a polymorphic tandem repeat in an intron of the human alanine:glyoxylate aminotransferase gene.

The autosomal recessive disease primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate amino-transferase (AGT). This paper concerns the identification, characterization and clinical use of an unusual discretely polymorphic tandem repeat sequence in the fourth intron of the human AGT gene (gene locus designation AGXT). In a random Caucasian population, three alleles could be clearly recognized that consisted of either 12 (type III), 17 (type II) or approximately 38 (type I) tandemly repeated copies of a highly conserved 29/32-bp sequence with frequencies of 33%, 7% and 60%, respectively. In a random Japanese population, the allelic frequencies were markedly different (i.e. 31%, 45% and 19%, respectively). In addition, a fourth allele was identified, consisting of approximately 32 repeats (type IV), with an allelic frequency of approximately 5% in Japanese. The repetitive sequence was similar to previously identified mammalian sequences with homology to the Epstein-Barr virus IR3 repetitive element involving a 12/15-bp region GCA(GGN)GGAGGAGGG within the repeat unit. This IR3-like sequence was interspersed with a 17-bp sequence with no similarity to any currently known repetitive element. The type I and type III alleles were judged to be equivalent to a previously identified TaqI polymorphism. Two polymorphisms previously shown to be associated with the peroxisome-to-mitochondrion mistargeting of AGT in PH1 (a C154-->T point substitution in exon 1 and a 74-bp duplication in intron 1) were found to segregate exclusively with the type I intron 4 polymorphism in Caucasians, but not in Japanese. The polymorphic nature of the intron 4 tandem repeats makes them of potential use in the prenatal diagnosis of PH1, especially when coupled with the exon 1 C154-->T substitution or intron 1 duplication polymorphisms. A PH1 family, in which a fetus had been predicted previously to be either normal or a carrier by AGT enzymic analysis of a fetal liver biopsy, but who had been shown to be only partially informative with respect to the C154-->T/intron 1 polymorphisms, was analysed retrospectively. The family was completely informative for the intron 4 tandem repeat polymorphism and the carrier status of the fetus was confirmed.

[Molecular pathology of type 1 primary hyperoxaluria]. / Pathologie moléculaire de l'hyperoxalurie primitive de type 1.

Type 1 is the most common form of primary hyperoxaluria, also called oxalosis when systemic involvement has occurred. This recessive autosomal inherited inborn error of metabolism is characterized by a defect of alanine: glyoxylate aminotransferase (AGT), which is a specific liver enzyme. This protein is responsible for glyoxylate detoxification only when it is located in the peroxisome. The clinical and biochemical phenotypes are neither correlated with the residual catalytic activity of AGT nor with its immunoreactivity. Most patients display less than 2% catalytic activity (enz-) or no immunoreactive protein (crm-); peroxisome-to-mitochondrion mistargeting is the main feature of patients crm+/enz+ or crm+/enz-. The cDNA and genomic DNA have been cloned and sequenced and the gene has been located on the long arm of chromosome 2 in the q36-37 region. Three polymorphisms have been identified which are preferentially associated, leading to two alleles; six point mutations have been currently reported.

Primary hyperoxaluria type 1 and peroxisome-to-mitochondrion mistargeting of alanine:glyoxylate aminotransferase.

Under the influence of dietary selection pressure, the intracellular compartmentalization of alanine:glyoxylate aminotransferase (AGT) has changed on many occasions during the evolution of mammals. In some mammals, AGT is peroxisomal in others it is mainly mitochondrial while in yet others it is more-or-less equally divided between both organelles. Although in normal human liver AGT is usually found exclusively within the peroxisomes, in some individuals a small proportion (approximately 5%) is found also in the mitochondria. This apparently trivial intracellular redistribution of AGT is caused by the presence of a Pro11Leu polymorphism which allows the N-terminus of AGT to fold into a conformation (ie a positively-charged amphiphilic alpha-helix) which functions as a mitochondrial targeting sequence. In one third of patients with the autosomal recessive disease primary hyperoxaluria type 1, there is a further redistribution of AGT so that the great majority (approximately 90%) is located in the mitochondria and only a small minority (10%) in the peroxisomes. AGT cannot fulfil its proper metabolic role in human liver (ie glyoxylate detoxification) when located in the mitochondria. The erroneous compartmentalization is due to the presence of a Gly170Arg mutation superimposed upon the Pro11Leu polymorphism. The Gly170Arg mutation appears to have no direct effect on mitochondrial targeting and is predicted to enhance mitochondrial import of AGT by interfering with its peroxisomal targeting and/or import. The mitochondrial targeting sequence generated by the Pro11Leu polymorphism is not homologous to that found in the AGT of other mammals which localise AGT within the mitochondria normally. The identity of the peroxisomal targeting sequence in AGT is unknown, but the Gly170Arg mutation is found in a highly conserved region of the protein which might be involved in some aspects of the peroxisomal import pathway for AGT.

Alanine glyoxylate aminotransferase deficiency: biochemical and molecular genetic lessons from the study of a human disease.

The decision to treat a patient with primary hyperoxaluria type 1 (PHI) by combined liver and kidney transplantation, the former to correct the metabolic lesion which was then thought to be deficiency of cytoplasmic 2-oxoglutarate:glyoxylate carboligase, and the latter to replace the organ which is destroyed, provided an opportunity to investigate the disease by modern biochemical methods. It was shown that 2-oxoglutarate:glyoxylate carboligase (the first decarboxylating component of 2-oxoglutarate dehydrogenase) is entirely mitochondrial so that deficiency of a cytoplasmic form of this enzyme could not be the cause of PHI. The deficient enzyme proved to be hepatic peroxisomal alanine:glyoxylate aminotransferase (AGT). The disease can be diagnosed enzymologically on percutaneous liver biopsies and this is possible for the fetus in utero. There are four types of genetically determined heterogeneity in PHI:(1) responsiveness and non-responsiveness to pharmacological doses of pyridoxine, in terms of an effect on the rate of oxalate production; (2) the presence or absence of residual catalytic AGT activity; (3) CRM+ and CRM-variants; (4) locational variation by virtue of which the enzyme (AGT) is mitochondrial and not peroxisomal. About one third of patients with PHI have residual AGT activity and at least a large proportion of these have mitochondrial and not peroxisomal AGT. The molecular features which guide peroxisomal and mitochondrial enzymes from their sites of synthesis into the appropriate organelle are reviewed and the possibilities for genetic variation in the relevant parts of the AGT molecule are discussed. The gene directing the synthesis of AGT has been cloned and sequenced, as has the AGT cDNA from a patient with mitochondrial AGT. Three point mutations causing amino acid substitution in the predicted AGT protein sequence have been identified: proline----leucine at residue 11, glycine----arginine at residue 170 and isoleucine----methionine at residue 340. The present evidence based on screening PHI patients and control subjects suggest that the substitution at residue 11, which cosegregates with that at residue 340, generates an amphiphilic alpha-helix which resembles mitochondrial targeting sequences but that misrouting of all the newly synthesized AGT into mitochondria requires the substitution at residue 170 which may act by impeding the entry of the enzyme into peroxisomes. The recognition of enzyme locational heterogeneity in PHI due to mutations affecting leader sequences should encourage a search for similar metabolic lesions in other inborn errors of metabolism affecting peroxisomal and/or mitochondrial enzymes.

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.

Combined liver-kidney and isolated liver transplantations for primary hyperoxaluria type 1: the European experience. The European Study Group on Transplantation in Hyperoxaluria Type 1.

The data provided by 14 European centres concerning 22 combined liver-kidney and two isolated liver grafts performed in primary hyperoxaluria type 1 (PH1) were discussed at a workshop which drew the following main conclusions: 1. In end-stage renal failure due to PH1 1-year kidney graft survival rate is far better after combined liver-kidney transplantation than after kidney transplantation alone. This may be due to enhanced renal graft tolerance induced by the simultaneously grafted liver, in addition to the reduced risk of oxalate-induced damage to the kidney graft because the oxalate overproduction has been corrected. 2. Prolonged dialysis using conventional regimes gives rise to extensive systemic oxalosis, especially oxalate osteopathy, which leads to long-lasting excretion of large amounts of oxalate even after oxalate synthesis has been normalised by liver-kidney transplantation, with the risk of jeopardising the success of the kidney graft. In addition, oxalate arteriopathy may endanger the recipient's life. 3. Patients whose GFR is in the range of 25-60 ml/min per 1.73 m2 should be followed up closely, with sequential assessments based on the rate of loss of overall renal function and the plasma and urine oxalate values. An isolated liver transplantation should be considered once the disease has been shown to be following an aggressive course. If this strategy is not followed, planning for an elective liver-kidney graft should begin when GFR decreases to about 25 ml/min per 1.73 m2 and the operation should be as soon as possible. 4. As orthotopic liver transplantation involves the removal of the recipient's biochemically defective but otherwise normal liver, the diagnosis of PH1 should be unequivocally established in every case by the measurement of alanine: glyoxylate aminotransferase enzyme activity in a preoperative liver biopsy.

Primary hyperoxaluria type I.

Primary hyperoxaluria type I is a metabolic disorder caused by the deficiency of the peroxisomal alanine:glyoxylate aminotransferase. The disease is inherited as an autosomal recessive trait. The clinical course is outlined based on data from 330 published cases. Diagnostic cornerstones are clinical parameters, urinary excretion of oxalate and glycolate, and the determination of enzyme activity in liver tissue. Principles of conservative treatment, e.g. volume load and pyridoxine substitution, are described as well as experience with different modes of dialysis and transplantation. Kidney transplantation is associated with a high rate of recurrence of the original disease despite excellent management resulting in many instances in early graft loss. Liver transplantation offers the possibility to correct the metabolic defect and to prevent the progression of crystal deposition in the body.

[Adult type I primary hyperoxaluria: 2 cases confirmed by liver biopsy at end-stage renal insufficiency]. / Hyperoxalurie primitive de type I de l'adulte: deux cas confirmés par biopsie hépatique au stade d'insuffisance rénale terminale.

It can be difficult to distinguish between primary hyperoxaluria at end-stage renal failure and secondary oxalosis, all the more as primary hyperoxaluria can be latent for a long time and occur at a late stage. A 57 year-old woman, without family nor personal history of urolithiasis, receives regular hemodialysis for a renal failure discovered at end-stage. Eighteen months later, calcium oxalate deposits appear in the skin, bone marrow and both kidneys, suggesting secondary oxalosis. An other 57 year-old woman presents a chronic renal failure due to bilateral urolithiasis, whose surgery has caused a dramatic decrease of renal function requiring regular hemodialysis. Because of apparition of severe bone alterations, a parathyroidectomy is realized, and because of calcium oxalate deposition in the skin and bone marrow, primary hyperoxaluria is suspected. In both observations, the enzyme activity determination in a liver biopsy gives the diagnosis of primary hyperoxaluria.

Fetal liver alanine: glyoxylate aminotransferase and the prenatal diagnosis of primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the hepatic peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT, EC 2.6.1.44) (Danpure and Jennings, FEBS Lett., 201, 20-24, 1986). The activity of AGT has been measured in fetal livers of gestational age 14-21 weeks. Activity increases up to 17 weeks and then levels off between 17 and 21 weeks. At this time, the mean AGT activity is about 30 per cent of the mean normal postnatal level. As in adult liver, the AGT enzyme activity and the AGT immunoreactive protein are peroxisomal. Prenatal diagnosis has been performed by measuring AGT enzyme activity and immunoreactive AGT protein on liver biopsies from two fetuses at risk for primary hyperoxaluria type 1. One was unaffected and one was affected.