Publications

• Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1.

  Aponte J.L., Sega G.A., Hauser L.J., Dhar M.S., Withrow C.M., Carpenter D.A., Rinchik E.M., Culiat C.T., Johnson D.K.

  Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic fai ... >> More

  Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah(6287SB) allele is a missense mutation in exon 6, and Fah(5961SB) is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah(6287SB) and Fah(5961SB) mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah(5961SB) and Fah(6287SB) as mouse models for acute and chronic forms of human HT1, respectively. << Less

  Proc. Natl. Acad. Sci. U.S.A. 98:641-645(2001) [PubMed] [EuropePMC]

• Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitor.

  Bateman R.L., Bhanumoorthy P., Witte J.F., McClard R.W., Grompe M., Timm D.E.

  Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing th ... >> More

  Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a K(i) of 85 microM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-A resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg(2+) bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors. << Less

  J. Biol. Chem. 276:15284-15291(2001) [PubMed] [EuropePMC]

• Crystal structure and mechanism of a carbon-carbon bond hydrolase.

  Timm D.E., Mueller H.A., Bhanumoorthy P., Harp J.M., Bunick G.J.

  <h4>Background</h4>Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mecha ... >> More

  <h4>Background</h4>Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mechanism. Carbon-carbon hydrolysis reactions are essential for the human metabolism of aromatic amino acids. FAH deficiency causes the fatal metabolic disease hereditary tyrosinemia type I. Carbon-carbon bond hydrolysis is also important in the microbial metabolism of aromatic compounds as part of the global carbon cycle.<h4>Results</h4>The FAH crystal structure has been determined by rapid, automated analysis of multiwavelength anomalous diffraction data. The FAH polypeptide folds into a 120-residue N-terminal domain and a 300-residue C-terminal domain. The C-terminal domain defines an unusual beta-strand topology and a novel 'mixed beta-sandwich roll' structure. The structure of FAH complexed with its physiological products was also determined. This structure reveals fumarate binding near the entrance to the active site and acetoacetate binding to an octahedrally coordinated calcium ion located in close proximity to a Glu-His dyad.<h4>Conclusions</h4>FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1. << Less

  Structure 7:1023-1033(1999) [PubMed] [EuropePMC]

• Characterization of the human fumarylacetoacetate hydrolase gene and identification of a missense mutation abolishing enzymatic activity.

  Labelle Y., Phaneuf D., Leclerc B., Tanguay R.M.

  Hereditary tyrosinemia type 1 is an autosomal recessive disease caused by a deficiency of the last enzyme in the catabolic pathway of tyrosine, fumarylacetoacetate hydrolase (FAH). To analyze the mutations involved in this disease, and as a first step towards elucidating the mechanisms regulating ... >> More

  Hereditary tyrosinemia type 1 is an autosomal recessive disease caused by a deficiency of the last enzyme in the catabolic pathway of tyrosine, fumarylacetoacetate hydrolase (FAH). To analyze the mutations involved in this disease, and as a first step towards elucidating the mechanisms regulating the transcription of the FAH gene, we have isolated and characterized the human gene coding for FAH. The gene contains 14 exons and spans approximately 35 kilobases of DNA. The 5' end of the gene is highly GC-rich, and eleven putative binding sites for the transcription factor Sp 1 were identified in the proximal region of the promoter. We investigated the molecular basis of FAH deficiency in a hereditary tyrosinemia type 1 patient whose liver FAH showed a very low enzymatic activity. Sequencing of the liver FAH cDNA of the patient revealed a C to A transversion in the FAH mRNA, which predicted the replacement of an alanine (A) residue with an aspartic acid (D) residue at position 134 (A134D) of the amino acid sequence of the corresponding protein. Direct sequencing of genomic DNA indicated that the patient was heterozygous for the A134D mutation. The allele that does not carry the A134D mutation was expressed at a very low level in the liver of the patient. Expression of the mutant allele in CV-1 cells confirmed that the A134D mutation was responsible for the lack of enzymatic activity in the liver of the patient. << Less

  Hum. Mol. Genet. 2:941-946(1993) [PubMed] [EuropePMC]