Maleylacetoacetate isomerase (MAAI/GSTZ)-deficient mice reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism

Abstract

In mammals, the catabolic pathway of phenylalanine and tyrosine is found in liver (hepatocytes) and kidney (proximal tubular cells). There are well-described human diseases associated with deficiencies of all enzymes in this pathway except for maleylacetoacetate isomerase (MAAI), which converts maleylacetoacetate (MAA) to fumarylacetoacetate (FAA). MAAI is also known as glutathione transferase zeta (GSTZ1). Here, we describe the phenotype of mice with a targeted deletion of the MAAI (GSTZ1) gene. MAAI-deficient mice accumulated FAA and succinylacetone in urine but appeared otherwise healthy. This observation suggested that either accumulating MAA is not toxic or an alternate pathway for MAA metabolism exists. A complete redundancy of MAAI could be ruled out because substrate overload of the tyrosine catabolic pathway (administration of homogentisic acid, phenylalanine, or tyrosine) resulted in renal and hepatic damage. However, evidence for a partial bypass of MAAI activity was also found. Mice doubly mutant for MAAI and fumarylacetoacetate hydrolase (FAH) died rapidly on a normal diet, indicating that MAA could be isomerized to FAA in the absence of MAAI. Double mutants showed predominant renal injury, indicating that this organ is the primary target for the accumulated compound(s) resulting from MAAI deficiency. A glutathione-mediated isomerization of MAA to FAA independent of MAAI enzyme was demonstrated in vitro. This nonenzymatic bypass is likely responsible for the lack of a phenotype in nonstressed MAAI mutant mice.

Figures

FIG. 1.

Catabolic pathway of tyrosine.

FIG. 2.

(A) Schematic representation of MAAI knockout. (B) Southern blot of wild-type (+/+) and heterozygote (+/−) DNA. The membrane was hybridized with the external probe shown in panel A. (C) Western blot with liver protein extracts from wild-type (+/+), heterozygote (+/−), and mutant (−/−) mice performed with MAAI antibody.

FIG. 3.

Gas chromatography of urine organic acids of wild-type, MAAI mutant, and MAAI/FAH double-mutant mice. Urine from three mice was pooled to avoid individual and time differences. FA, fumarylacetone; MA, maleylacetone.

FIG. 4.

Liver RNA Northern blot from mice on regular or high-phenylalanine (20 mg/ml in drinking water) diet. Lanes: 1 and 2, wild type on regular diet; 3 to 5, MAAI mutant on regular diet; 6, FAH mutant on regular diet without NTBC for 6 days; 7, FAH mutant on regular diet without NTBC for 2 weeks; 8 to 10, MAAI/FAH double mutant on regular diet without NTBC for 6 days; 11 and 12, wild type on high-phenylalanine diet; 13 and 14, mutant mice on high-phenylalanine diet; 15, MAAI heterozygote on high-phenylalanine diet; 16, wild type on high-phenylalanine diet plus NTBC; 17; MAAI mutant on high-phenylalanine diet plus NTBC; 18, mice 18 months old on regular diet; 19, MAAI mutant treated with DEM plus HGA; 20, wild type treated with DEM plus HGA.

FIG. 5.

(A) Sensitivity to HGA (intraperitoneal injections). Ten mice were injected for each point. (B) Mouse weight variation on phenylalanine (20 mg/ml) in drinking water. (C) Mouse weight variation on bean diet. Diet was 25% (each) soy beans, peas, lentils, and white beans.

FIG. 6.

Schematic representation of metabolites in the distal tyrosine catabolic pathway. FA, fumaric acid; AA, acetoacetate. Arrows with solid lines indicate active reactions, and the thickness of the arrow correlates to the relative quantities. Arrows with dashed lines indicate blocked reactions. (A) Normal tyrosine metabolism distal to HGA. MAA is converted to FAA by MAAI in a GSH-dependent reaction. FAA is hydrolyzed to FA and AA by FAH. (B) MAAI deficiency with normal diet. Modest amounts of MAA are formed and then isomerized to FAA by a nonenzymatic bypass reaction. Small amounts of MAA accumulate, leave the hepatocyte, and are converted to FAA and SA, which appear in the urine. (C) MAAI deficiency on a high-phenylalanine diet. Large amounts of MAA are produced from the dietary load, thus overwhelming the nonenzymatic bypass of MAAI. Free MAA accumulates (MAA∗) and results in cytotoxicity. (D) FAH/MAI double mutants on a regular diet. MAA is converted to FAA by the nonenzymatic bypass. MAA and FAA accumulate (MAA∗ and FAA∗) and are cytotoxic. (E) MAI deficiency with GSH depletion. MAA cannot be converted to FAA, because both MAI and the nonenzymatic pathway are defective. MAA accumulates (MAA∗) and is cytotoxic.

FIG. 7.

MAAI activity in MAAI mutant mouse liver. Activity was restored only when 2 μg of purified recombinant MAAI or 10 mM GSH was added to the reaction mixture. Gaps indicate the time of the addition.