Maleylacetoacetate Isomerase Research Articles

Age-Dependent Kinetics and Metabolism of Dichloroacetate: Possible Relevance to Toxicity

Dichloroacetate (DCA) is an investigational drug for certain metabolic diseases. It is biotransformed principally by the zeta-1 family isoform of glutathione transferase (GSTz1), also known as maleylacetoacetate isomerase (MAAI), which catalyzes the penultimate step in tyrosine catabolism. DCA causes a reversible peripheral neuropathy in several species, including humans. However, recent clinical trials indicate that adults are considerably more susceptible to this adverse effect than children. We evaluated the kinetics and biotransformation of DCA and its effects on tyrosine metabolism in nine patients treated for 6 months with 25 mg/kg/day and in rats treated for 5 days with 50 mg/kg/day. We also measured the activity and expression of hepatic GSTz1/MAAI. Chronic administration of DCA causes a striking age-dependent decrease in its plasma clearance and an increase in its plasma half-life in patients and rats. Urinary excretion of unchanged DCA in rats increases with age, whereas oxalate, an end product of DCA metabolism, shows the opposite trend. Low concentrations of monochloroacetate (MCA), which is known to be neurotoxic, increase as a function of age in the urine of dosed rats. MCA was detectable in plasma only of older animals. Hepatic GSTz1/MAAI-specific activity was inhibited equally by DCA treatment among all age groups, whereas plasma and urinary levels of maleylacetone, a natural substrate for this enzyme, increased with age. We conclude that age is an important variable in the in vivo metabolism and elimination of DCA and that it may account, in part, for the neurotoxicity of this compound in humans and other species.

Crystal Structures and Site-directed Mutagenesis of a Mycothiol-dependent Enzyme Reveal a Novel Folding and Molecular Basis for Mycothiol-mediated Maleylpyruvate Isomerization

Mycothiol (MSH) is the major low molecular mass thiols in many Gram-positive bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. The physiological roles of MSH are believed to be equivalent to those of GSH in Gram-negative bacteria, but current knowledge of MSH is limited to detoxification of alkalating chemicals and protection from host cell defense/killing systems. Recently, an MSH-dependent maleylpyruvate isomerase (MDMPI) was discovered from C. glutamicum, and this isomerase represents one example of many putative MSH-dependent enzymes that take MSH as cofactor. In this report, fourteen mutants of MDMPI were generated. The wild type and mutant (H52A) MDMPIs were crystallized and their structures were solved at 1.75 and 2.05 A resolution, respectively. The crystal structures reveal that this enzyme contains a divalent metal-binding domain and a C-terminal domain possessing a novel folding pattern (alphabetaalphabetabetaalpha fold). The divalent metal-binding site is composed of residues His52, Glu144, and His148 and is located at the bottom of a surface pocket. Combining the structural and site-directed mutagenesis studies, it is proposed that this surface pocket including the metal ion and MSH moiety formed the putative catalytic center.

Enzymes of tyrosine catabolism in Arabidopsis thaliana

Tyrosine catabolism is an essential pathway in animals, but its role in plants is unclear. The first steps of tyrosine degradation lead to the formation of homogentisate. In animals this is then sequentially acted on by homogentisate dioxygenase (HGO), maleylacetoacetate isomerase (MAAI) and fumarylacetoacetate hydrolase (FAH) to generate fumarate and acetoacetate. In plants, homogentisate is used to generate the essential redox metabolites tocopherol and plastoquinone, which effectively act as an alternative metabolic fate for tyrosine. Having determined that a zeta class glutathione transferase from Arabidopsis thaliana is a functional MAAI, we have looked for evidence that the mammalian degradation pathway could also operate in plants. Based on array and quantitative PCR experiments, the A. thaliana homologues AtHGO, AtMAAI and AtFAH could be shown to be expressed, with AtHGO and AtMAAI showing evidence of co-regulation. cDNAs encoding AtHGO, AtMAAI and AtFAH were cloned in Escherichia coli and shown to represent a fully functional catabolic pathway when combined in vitro. The significance of this pathway, including increased transcription of the associated enzymes in senescing tissue, compartmentalisation and impact on flux into synthesis of Vitamin E and other tocopherols of biotechnological interest is discussed.

Evaluation of dichloroacetate treatment in a murine model of hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease severely affecting liver and kidney and is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Administration of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) improves the HT1 phenotype but some patients do not respond to NTBC therapy. The objective of the present study was to evaluate whether administration of dichloroacetate, an inhibitor of maleyl acetoacetate isomerase (MAAI) to FAH-knockout mice could prevent acute pathological injury caused by NTBC withdrawal. DCA (0.5 and 5 g/L) was given in combination with a standard diet or with a tyrosine-restricted diet. With the low-tyrosine diet body weight loss and most of hepatic and renal injuries were prevented regardless the DCA dose. The administration of DCA with a standard diet did not prevent damage nor the oxidative stress response nor the AFP induction seen in FAH-knockout mice. DCA was shown to inhibit hepatic MAAI activity to 86% (0.5 g/L) and 94% (5 g/L) of untreated wild-type mice. Interestingly, FAH −/− mice deprived of NTBC (NTBC-OFF) and NTBC-treated FAH-knockout mice had similar low hepatic MAAI activity levels, corresponding to 10–20% of control. Thus the failure of DCA treatment in FAH −/− mice seems to be attributed to the residual MAAI activity, high enough to lead to FAA accumulation and HT1 phenotype.

A Mechanistic Investigation of the Thiol−Disulfide Exchange Step in the Reductive Dehalogenation Catalyzed by Tetrachlorohydroquinone Dehalogenase

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachloro- and trichlorohydroquinone to give 2,6-dichlorohydroquinone in the pathway for degradation of pentachlorophenol by Sphingobium chlorophenolicum. Previous work has suggested that this enzyme may have originated from a glutathione-dependent double bond isomerase such as maleylacetoacetate isomerase or maleylpyruvate isomerase. While some of the elementary steps in these two reactions may be similar, the final step in the dehalogenation reaction, a thiol-disulfide exchange reaction that removes glutathione covalently bound to Cys13, certainly has no counterpart in the isomerization reaction. The thiol-disulfide exchange reaction does not appear to have been evolutionarily optimized. There is little specificity for the thiol; many thiols react at high rates. TCHQ dehalogenase binds the glutathione involved in the thiol-disulfide exchange reaction very poorly and does not alter its pK(a) in order to improve its nucleophilicity. Remarkably, single-turnover kinetic studies show that the enzyme catalyzes this step by approximately 10000-fold. This high reactivity requires an as yet unidentified protonated group in the active site.

Mice Deficient in Glutathione Transferase Zeta/Maleylacetoacetate Isomerase Exhibit a Range of Pathological Changes and Elevated Expression of Alpha, Mu, and Pi Class Glutathione Transferases

Glutathione transferase zeta (GSTZ1-1) is the major enzyme that catalyzes the metabolism of alpha-halo acids such as dichloroacetic acid, a carcinogenic contaminant of chlorinated water. GSTZ1-1 is identical with maleylacetoacetate isomerase, which catalyzes the penultimate step in the catabolic pathways for phenylalanine and tyrosine. In this study we have deleted the Gstz1 gene in BALB/c mice and characterized their phenotype. Gstz1(-/-) mice do not have demonstrable activity with maleylacetone and alpha-halo acid substrates, and other GSTs do not compensate for the loss of this enzyme. When fed a standard diet, the GSTZ1-1-deficient mice showed enlarged liver and kidneys as well as splenic atrophy. Light and electron microscopic examination revealed multifocal hepatitis and ultrastructural changes in the kidney. The addition of 3% (w/v) phenylalanine to the drinking water was lethal for young mice (<28 days old) and caused liver necrosis, macrovesicular steatosis, splenic atrophy, and a significant loss of circulating leukocytes in older surviving mice. GSTZ1-1-deficient mice showed constitutive induction of alpha, mu, and pi class GSTs as well as NAD(P)H:quinone oxidoreductase 1. The overall response is consistent with the chronic accumulation of a toxic metabolite(s). We detected the accumulation of succinylacetone in the serum of deficient mice but cannot exclude the possibility that maleylacetoacetate and maleylacetone may also accumulate.

The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida.

Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

Pharmacologic or genetic ablation of maleylacetoacetate isomerase increases levels of toxic tyrosine catabolites in rodents

Dichloroacetate (DCA) is both an environmental contaminant and an investigational drug for diseases involving perturbed mitochondrial energetics. DCA is biotransformed to glyoxylate by maleylacetoacetate isomerase (MAAI). Previous studies have shown that DCA decreases MAAI activity in rat liver in a time- and dose-dependent manner and may target the protein for degradation in vivo. We now report that the MAAI protein is depleted in a time- and dose-dependent manner in the livers of Sprague–Dawley rats exposed to DCA. This decrease in protein expression is not mirrored by a decrease in the steady-state levels of MAAI mRNA, indicating that the depletion is exclusively a post-transcriptional event. We also investigated the pharmacokinetics of DCA in the recently developed MAAI knockout (MAAI-KO) mouse. MAAI-KO mice maintain high plasma and urine drug concentrations and do not biotransform DCA to monochloroacetate to a significant extent. Therefore, no alternative pathways for DCA clearance appear to exist in mice other than by MAAI-mediated biotransformation. DCA-naı̈ve MAAI-KO mice accumulate very high levels of the tyrosine catabolites maleylacetone and succinylacetone, and DCA exposure did not significantly increase the levels of these compounds. MAAI-KO mice also have high levels of fumarylacetone and normal levels of fumarate. These results demonstrate that pharmacologic or genetic ablation of MAAI cause potentially toxic concentrations of tyrosine intermediates to accumulate in mice and perhaps in other species.

Clarification of the role of key active site residues of glutathione transferase zeta/maleylacetoacetate isomerase by a new spectrophotometric technique.

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(+/-)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16-->Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the K(m) for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of alpha-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the alpha-halo acid substrates.

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

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.

Dichloroacetate toxicokinetics and disruption of tyrosine catabolism in B6C3F1 mice: dose–response relationships and age as a modifying factor

Dichloroacetate (DCA) is a rodent carcinogen commonly found in municipal drinking water supplies. Toxicokinetic studies have established that elimination of DCA is controlled by liver metabolism, which occurs by the cytosolic enzyme glutathione- S-transferase-zeta (GST-zeta). DCA is also a mechanism based inhibitor of GST-zeta, and a loss in GST-zeta enzyme activity occurs following repeated doses or prolonged drinking water exposures. GST-zeta is identical to an enzyme that is part of the tyrosine catabolism pathway known as maleylacetoacetate isomerase (MAAI). In this pathway, GST-zeta plays a critical role in catalyzing the isomerization of maleylacetoacetate to fumarylacetoacetate. Disruption of tyrosine catabolism has been linked to increased cancer risk in humans. We studied the elimination of i.v. doses of DCA to young (10 week) and aged (60 week) mice previously treated with DCA in their drinking water for 2 and 56 weeks, respectively. The diurnal change in blood concentrations of DCA was also monitored in mice exposed to three different drinking water concentrations of DCA (2.0, 0.5 and 0.05 g/l). Additional experiments measured the in vitro metabolism of DCA in liver homogenates prepared from treated mice given various recovery times following treatment. The MAAI activity was also measured in liver cytosol obtained from treated mice. Results indicated young mice were the most sensitive to changes in DCA elimination after drinking water treatment. The in vitro metabolism of DCA was decreased at all treatment rates. Partial restoration (∼65% of controls) of DCA elimination capacity and hepatic GST-zeta activity occurred after 48 h recovery from 14 d 2.0 g/l DCA drinking water treatments. Recovery from treatments could be blocked by interruption of protein synthesis with actinomycin D. MAAI activity was reduced over 80% in liver cytosol from 10-week-old mice. However, MAAI was unaffected in 60-week-old mice. These results indicate that in young mice, inactivation and re-synthesis of GST-zeta is a highly dynamic process and that exogenous factors that deplete or reduce GST-zeta levels will decrease DCA elimination and may increase the carcinogenic potency of DCA. As mice age, the elimination capacity for DCA is less affected by reduced liver metabolism and mice appear to develop some toxicokinetic adaptation(s) to allow elimination of DCA at rates comparable to naive animals. Reduced MAAI activity alone is unlikely to be the carcinogenic mode of action for DCA and may in fact, only be important during the early stages of DCA exposure.

GSTZ1d: a new allele of glutathione transferase zeta and maleylacetoacetate isomerase.

The zeta class glutathione transferases (GSTs) are known to catalyse the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA), and the biotransformation of dichloroacetic acid to glyoxylate. A new allele of human GSTZ1, characterized by a Thr82Met substitution and termed GSTZ1d, has been identified by analysis of the expressed sequence tag (EST) database. In European Australians, GSTZ1d occurs with a frequency of 0.16. Like GSTZ1b-1b and GSTZ1c-1c, the new isoform has low activity with dichloroacetic acid compared with GSTZ1a-1a. The low activity appears to be due to a high sensitivity to substrate inhibition. The maleylacetoacetate isomerase (MAAI) activity of all known variants was compared using maleylacetone as a substrate. Significant differences in activity were noted, with GSTZ1a-1a having a notably lower catalytic efficiency. The unusual catalytic properties of GSTZ1a-1a in both reactions suggest that its characteristic arginine at position 42 plays a significant role in the regulation of substrate access and/or product release. The different amino acid substitutions have been mapped on to the recently determined crystal structure of GSTZ1-1 to evaluate and explain their influence on function.

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.

Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach

The pathway for degradation of the xenobiotic pesticide pentachlorophenol in Sphingomonas chlorophenolica probably evolved in the past few decades by the recruitment of enzymes from two other catabolic pathways. The first and third enzymes in the pathway, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, may have originated from enzymes in a pathway for degradation of a naturally occurring chlorinated phenol. The second enzyme, a reductive dehalogenase, may have evolved from a maleylacetoacetate isomerase normally involved in degradation of tyrosine. This apparently recently assembled pathway does not function very well: pentachlorophenol hydroxylase is quite slow, and tetrachlorohydroquinone dehalogenase is subject to severe substrate inhibition.

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.

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 ∼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.

Comparative tyrosine degradation in Vibrio cholerae strains. The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell

The relationship between l-tyrosine catabolism and melanin formation was studied in the Vibrio cholerae strains ATCC 14035 and CECT 557. It is shown that both strains degrade l-tyrosine by the same pathway as eukaryotic cells, giving homogentisate as intermediate. ATCC 14035, an O1 strain, which is not able to grow using l-tyrosine as sole carbon and energy source, but it forms pyomelanin from homogentisate. The second strain, which is non-O1, is able to grow using l-tyrosine as sole carbon and energy source, but it does not form any pigment. Both strains contain all the enzymes involved in the l-tyrosine catabolism. The three late enzymes of the pathway, homogentisate oxygenase, maleylacetoacetate isomerase and fumarylacetoacetate hydrolase, are induced by l-tyrosine, but the degree of induction is much lower in the ATCC 14035 strain. Thus, the distal part of the pathway becomes the rate-limiting steps in the l-tyrosine catabolism, explaining homogentisate accumulation and pyomelanogenesis in this strain. It is proposed that V. cholerae might be a useful prokaryotic model to show that alkaptonuria and other diseases related to l-tyrosine metabolism could occur in animals even when no particular enzyme involved in that pathway is lacking.

Characterization and chromosome location of the gene GSTZ1 encoding the human Zeta class glutathione transferase and maleylacetoacetate isomerase

The Zeta class of cytosolic glutathione-S-transferases (GSTs) has recently been identified and spans a range of species from plants to humans. The cDNA and protein of a human member of this class have been previously characterised in our laboratory. This cDNA has also been described as maleylacetoacetate isomerase (MAAI), an enzyme of the phenylalanine catabolism pathway (Fernandez-Canon and Penalva, 1998). The present study has determined the structure and chromosome location of the gene encoding human GSTZ1/MAAI. The gene spans approximately 10.9 kb and is composed of 9 exons. Three intron positions of GSTZ1 were precisely conserved compared to the carnation and Caenorhabditis elegans Zeta GST genes. Fluorescent in situ hybridization mapped the gene to a single locus on chromosome 14q24.3, which is in agreement with an independent localization between the Genethon markers D14S263 and D14S67. The coding region of the gene differed from the GSTZ1 cDNA at two nucleotide positions in exon 3, resulting in Lys-32→Glu and Arg-42→ Gly substitutions. This gene structure information will allow analysis of the polymorphism in genomic DNA samples, and enables further investigations into genetic defects in this step of the phenylalanine catabolism pathway.

Characterization of a Fungal Maleylacetoacetate Isomerase Gene and Identification of Its Human Homologue

We have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway. We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene. maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways. Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli. Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulans strain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2, 4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency.

Maleylacetoacetate cis-trans isomerase: One-step double cis-trans isomerization of monomethyl muconate and the enzyme's probable role in benzene metabolism

Maleylacetoacetate cis-trans isomerase together with glutathione has been found to isomerize cis-trans isomers of monomethyl muconate. Isomerization about a single double bond and concerted double isomerization of the diene unit occurs. In addition to the variations in substrate structure previously identified the current results demonstrate that a cis,cis diene skeleton and a conjugated ester function are accepted by the enzyme. The present work and the fiding of trans,trans-muconic acid in the urine of benzene-fed mice ( M. M. Gad-El-Karim, V. M. Sadagopa Ramanujam, and M. S. Legator (1985) Xenobiotica 15, 211) suggest that maleylacetoacetate cis-trans isomerase may be responsible for the geometrical isomerization. However, cis,cis-muconaldehydic acid rather than cis,cis-muconic acid is suggested to be the early intermediate in benzene metabolism capable of rapid enzyme-catalyzed cis-trans isomerization.