Gene Structure, Chromosomal Location, and Expression Pattern of Maleylacetoacetate Isomerase

The gene for maleylacetoacetate isomerase (MAAI) (EC 5.2.1.2) was the last gene in the mammalian phenylalanine/tyrosine catabolic pathway to be cloned. We have isolated the human and murine genes and determined their genomic structure. The human gene spans a genomic region of approximately 10 kb, has 9 exons ranging from 50 to 528 bp in size, and was mapped to 14q24.3-14q31.1 using fluorescence in situ hybridization. The complete catabolic pathway of phenylalanine/tyrosine is normally restricted to liver and kidney, but the maleylacetoacetate isomerase gene is expressed ubiquitously. This suggests a possible second role for the MAAI protein different from phenylalanine/tyrosine catabolism. We have searched for mutations in the maleylacetoacetate isomerase gene in four cases of unexplained severe liver failure in infancy with clinical similarities to hereditary tyrosinemia type I (pseudotyrosinemia). Several amino acid changes were identified, but all were found to retain MAAI activity and thus represent protein polymorphisms. We conclude that MAAI deficiency is not a common cause of the pseudotyrosinemic phenotype.     

Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2

GSTZs [Zeta class GSTs (glutathione transferases)] are multifunctional enzymes that belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZs are known to function as MAAIs [MAA (maleylacetoacetate) isomerases], which play a role in tyrosine catabolism by catalysing the isomerization of MAA to FAA (fumarylacetoacetate). As tyrosine metabolism in plants differs from animals, the significance of GSTZ/MAAI is unclear. In rice (Oryza sativa L.), a major QTL (quantitative trait locus) for seedling cold tolerance has been fine mapped to a region containing the genes OsGSTZ1 and OsGSTZ2. Sequencing of tolerant (ssp. japonica cv. M-202) and sensitive (ssp. indica cv. IR50) cultivars revealed two SNPs (single nucleotide polymorphisms) in OsGSTZ2 that result in amino acid differences (I99V and N184I). Recombinant OsGSTZ2 containing the Val99 residue found in IR50 had significantly reduced activity on MAA and DCA (dichloroacetic acid), but the Ile184 residue had no effect. The distribution of the SNP (c.295A>G) among various rice accessions indicates a significant association with chilling sensitivity in rice seedlings. The results of the present study show that naturally occurring OsGSTZ2 isoforms differ in their enzymatic properties, which may contribute to the differential response to chilling stress generally exhibited by the two major rice subspecies.     

Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity

Maleylacetoacetate isomerase (MAAI), a key enzyme in the metabolic degradation of phenylalanine and tyrosine, catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate. Deficiencies in enzymes along the degradation pathway lead to serious diseases including phenylketonuria, alkaptonuria, and the fatal disease, hereditary tyrosinemia type I. The structure of MAAI might prove useful in the design of inhibitors that could be used in the clinical management of the latter disease. Here we report the crystal structure of human MAAI at 1.9 A resolution in complex with glutathione and a sulfate ion which mimics substrate binding. The enzyme has previously been shown to belong to the zeta class of the glutathione S-transferase (GST) superfamily based on limited sequence similarity. The structure of MAAI shows that it does adopt the GST canonical fold but with a number of functionally important differences. The structure provides insights into the molecular bases of the remarkable array of different reactions the enzyme is capable of performing including isomerization, oxygenation, dehalogenation, peroxidation, and transferase activity.     

Inhibition of glutathione S-transferase zeta and tyrosine metabolism by dichloroacetate: a potential unifying mechanism for its altered biotransformation and toxicity.

Dichloroacetate (DCA) inhibits its own metabolism and is converted to glyoxylate by glutathione S-transferase zeta (GSTz). GSTz is identical to maleylacetoacetate isomerase, an enzyme of tyrosine catabolism that converts maleylacetoacetate (MAA) to fumarylacetoacetate and maleylacetone (MA) to fumarylacetone. MAA and MA are alkylating agents. Rats treated with DCA for up to five days had markedly decreased hepatic GSTz activity and increased urinary excretion of MA. When dialyzed cytosol obtained from human liver was incubated with DCA, GSTz activity was unaffected. In contrast, DCA incubation inhibited enzyme activity in dialyzed hepatic cytosol from rats. Incubation of either rat or human hepatic cytosol with MA led to a dose dependent inhibition of GSTz. These data indicate that humans or rodents exposed to DCA may accumulate MA and/or MAA which inhibit(s) GSTz and, consequently, DCA biotransformation. Moreover, DCA-induced inhibition of tyrosine catabolism may account for the toxicity of this xenobiotic in humans and other species.     

Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.

Hereditary tyrosinemia type I is the most severe metabolic disease of the tyrosine catabolic pathway mainly affecting the liver. It is caused by deficiency of fumarylacetoacetate hydrolase, which prevents degradation of the toxic metabolite fumarylacetoacetate (FAA). We report here that FAA induces common effects (i.e., cell cycle arrest and apoptosis) in both human (HepG2) and rodent (Chinese hamster V79) cells, effects that seem to be temporally related. Both the antiproliferative and apoptosis-inducing activities of FAA are dose dependent and enhanced by glutathione (GSH) depletion with L-buthionine-(S,R)-sulfoximine (BSO). Short treatment (2 h) with 35 microM FAA/+BSO or 100 microM FAA/-BSO induced a transient cell cycle arrest at the G2/M transition (20% and 37%, respectively) 24 h post-treatment. In cells treated with 100 microM FAA/-BSO, an inactivation, followed by a rapid over-induction of cyclin B-dependent kinase occurred, which peaked 24 h post-treatment. Maximum levels of caspase-1 and caspase-3 activation were detected at 3 h and 32 h, respectively, whereas release of mitochondrial cytochrome c was maximal at 24-32 h post-treatment. The G2/M peak declined 24 h later, concomitantly with the appearance of a sub-G1, apoptotic population showing typical nucleosomal-sized DNA fragmentation and reduced mitochondrial transmembrane potential (Deltapsi(m)). These events were prevented by the general caspase inhibitor z-VAD-fmk, whereas G2/M arrest and subsequent apoptosis were abolished by GSH-monoethylester or N-acetylcysteine. Other tyrosine metabolites, maleylacetoacetate and succinylacetone, had no antiproliferative effects and induced only very low levels of apoptosis. These results suggest a modulator role of GSH in FAA-induced cell cycle disturbance and apoptosis where activation of cyclin B-dependent kinase and caspase-1 are early events preceding mitochondrial cytochrome c release, caspase-3 activation, and Deltapsi(m) loss. -Jorquera, R., Tanguay, R. M. Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.     

家蚕谷胱甘肽-S-转移酶基因BmGSTz1的克隆、序列分析及组织表达特征

谷胱甘肽-S-转移酶(GSTs)是一个功能广泛的超基因家族, 其中Zeta家族在动物、植物和细菌中均有分布。在哺乳动物中, Zeta GSTs具有马来酰乙酰乙酸异构酶（MAAI）活性, 参与苯丙氨酸/酪氨酸的代谢过程。本研究对家蚕Bombyx mori基因组中预测的GST基因（BmGSTz1）进行了表达序列标签的搜索, 经拼接后获得一条含有3′和5′非翻译区在内的长度为1 239 bp 的cDNA序列, 其3′端含有AATAAA加尾信号。BmGSTz1基因含有4个内含子, 外显子/内含子边界均符合GT-AG 规则。经TA克隆证实, BmGSTz1基因编码区序列全长648 bp, 共编码215个氨基酸。BmGSTz1推定的分子量为24.8 kD, 等电点pI为8.06。BmGSTz1与其他昆虫和哺乳动物GSTz1的氨基酸序列高度保守, 进化分析表明家蚕BmGSTz1与黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、意大利蜜蜂Apis mellifera和赤拟谷盗Tribolium castaneum的GSTz1形成1∶1∶1∶1∶1的直系同源关系。RT-PCR和基因芯片数据表明BmGSTz1在家蚕5龄第3天幼虫各组织中都有表达。序列和组织表达特征分析结果提示家蚕BmGSTz1可能具有MAAI活性, 这将为进一步深入研究BmGSTz1基因的功能提供参考。     

Quantitative analysis of DNA binding by the Escherichia coli arginine repressor

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed.     

Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol

Tetrachlorohydroquinone dehalogenase catalyzes the replacement of chlorine atoms on tetrachlorohydroquinone and trichlorohydroquinone with hydrogen atoms during the biodegradation of pentachlorophenol by Sphingomonas chlorophenolica. The sequence of the active site region of tetrachlorohydroquinone dehalogenase is very similar to those of the corresponding regions of maleylacetoacetate isomerases, enzymes that catalyze the glutathione-dependent isomerization of a cis double bond in maleylacetoacetate to the trans configuration during the catabolism of phenylalanine and tyrosine. Furthermore, tetrachlorohydroquinone dehalogenase catalyzes the isomerization of maleylacetone (an analogue of maleylacetoacetate) at a rate nearly comparable to that of a bona fide bacterial maleylacetoacetate isomerase. Since maleylacetoacetate isomerase is involved in a common and presumably ancient pathway for catabolism of tyrosine, while tetrachlorohydroquinone dehalogenase catalyzes a more specialized reaction, it is likely that tetrachlorohydroquinone dehalogenase arose from a maleylacetoacetate isomerase. The substrates and overall transformations involved in the dehalogenation and isomerization reactions are strikingly different. This enzyme provides a remarkable example of Nature's ability to recruit an enzyme with a useful structural scaffold and elaborate upon its basic catalytic capabilities to generate a catalyst for a newly needed reaction.     

Degradation of homogentisate by strains of Bacillus and Moraxella.

The metabolic pathways whereby strains of Moraxella and Bacillus degrade homogentisate (2,5-dihydroxyphenylacetate) are delineated. The Moraxella (strain OA3) is shown to degrade homogentisate via the pathway previously described in liver: homogentisate is cleaved by a 1,2-dioxygenase (E.C 1.13.11.5) yielding maleylacetoacetate which is isomerized by a GSH-dependent isomerase to fumarylacetoacetate before hydrolysis to acetoacetate and fumarate. A strain of Bacillus (B11c) is shown to catabolize homogentisate via a previously undescribed version of the above sequence: homogentisate is cleaved by a 1,2-dioxygenase (E.C 1.13.11.5) yielding maleylacetoacetate which is hydrolyzed directly to acetoacetate and maleate.     

Degradation of 4-hydroxyphenylacetate by Xanthobacter 124X

Xanthobacter 124X when grom on 4-hydroxyphenylacetate was able to hydroxylate this compound yielding homogenisate. Ring fission of this latter compound gave maleylacetoacetate which was isomerized to fumarylacetoacetate. The isomerase involved resembled maleylacetoacetate isomerases in Gram-negative bacteria in that glutathione was required for activity. Fumarate and acetoacetate were both detected as products of the hydrolysis of fumarylacetoacetate.     

Loss of Sprouty2 partially rescues renal hypoplasia and stomach hypoganglionosis but not intestinal aganglionosis in Ret Y1062F mutant mice

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.     

Regulation of Aromatic Amino Acid Biosynthesis and Production of Tyrosine and Phenylalanine m Brevibacterium flavum

In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy-D-arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine(mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed.     

C/EBPalpha-dependent induction of glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI) expression during the differentiation of mouse fibroblasts into adipocytes

Western blot analysis of 3T3-L1 adipocyte proteins using an anti-C/EBPalpha antibody detected a 24kD polypeptide in addition to the expected 42 and 30kD isoforms of C/EBPalpha. Mass spectrometric sequencing of the protein following its purification by HPLC and preparative 2D gel electrophoresis identified it as glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI). Expression of GSTzeta/MAAI mRNA and protein was induced during the terminal phase of adipogenesis in 3T3-L1 preadipocytes. Ectopic expression of PPARgamma2 in NIH-3T3 fibroblasts exposed to insulin and troglitazone-induced perilipin production, but was incapable of activating GSTzeta/MAAI unless C/EBPalpha was also expressed. Similarly, ectopic expression of C/EBPalpha in PPARgamma +/- or PPARgamma -/- MEFs demonstrated that the C/EBPalpha-dependent induction of GSTzeta/MAAI production was dependent on expression of endogenous PPARgamma. These data suggest a role for GSTzeta/MAAI in mature adipocytes that may be responsive to the thiazolidinedione class of insulin sensitizing PPARgamma ligands.     

The Caenorhabditis elegans K10C2.4 gene encodes a member of the fumarylacetoacetate hydrolase family: a Caenorhabditis elegans model of type I tyrosinemia

In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a five-step tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied; however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia.     

A missense mutation (Q279R) in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation

Background  

Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

GSTZ1 sensitizes hepatocellular carcinoma cells to sorafenib-induced ferroptosis via inhibition of NRF2/GPX4 axis

Increasing evidence supports that ferroptosis plays an important role in tumor growth inhibition. Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, has been shown to induce ferroptosis in hepatocellular carcinoma (HCC). However, some hepatoma cell lines are less sensitive to sorafenib-induced ferroptotic cell death. Glutathione S-transferase zeta 1 (GSTZ1), an enzyme in the catabolism of phenylalanine, suppresses the expression of the master regulator of cellular redox homeostasis nuclear factor erythroid 2-related factor 2 (NRF2). This study aimed to investigate the role and underlying molecular mechanisms of GSTZ1 in sorafenib-induced ferroptosis in HCC. GSTZ1 was significantly downregulated in sorafenib-resistant hepatoma cells. Mechanistically, GSTZ1 depletion enhanced the activation of the NRF2 pathway and increased the glutathione peroxidase 4 (GPX4) level, thereby suppressing sorafenib-induced ferroptosis. The combination of sorafenib and RSL3, a GPX4 inhibitor, significantly inhibited GSTZ1-deficient cell viability and promoted ferroptosis and increased ectopic iron and lipid peroxides. In vivo, the combination of sorafenib and RSL3 had a synergic therapeutic effect on HCC progression in Gstz1^−/− mice. In conclusion, this finding demonstrates that GSTZ1 enhanced sorafenib-induced ferroptosis by inhibiting the NRF2/GPX4 axis in HCC cells. Combination therapy of sorafenib and GPX4 inhibitor RSL3 may be a promising strategy in HCC treatment.Subject terms: Cancer therapeutic resistance, Cancer therapeutic resistance