Phosphoryl Transferase Research Articles

An RNA Interference (RNAi) Toolkit and Its Utility for Functional Genetic Analysis of Leishmania (Viannia).

RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis (Lbr). We developed vectors facilitating generation of long-hairpin or "stem-loop" (StL) RNAi knockdown constructs, using GatewayTM site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as quinonoid dihydropteridine reductase (QDPR), a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful 'hypomorphic' approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a finding that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

Globally correlated conformational entropy underlies positive and negative cooperativity in a kinase\u2019s enzymatic cycle

Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studied the conformational landscape of the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous phosphoryl transferase involved in a myriad of cellular processes. Along the enzymatic cycle, the kinase exhibits positive and negative cooperativity for substrate and nucleotide binding and product release. We found that globally coordinated changes of conformational entropy activated by ligand binding, together with synchronous and asynchronous breathing motions of the enzyme, underlie allosteric cooperativity along the kinase’s cycle.

P3.03-005 Inhibition of PRMT5 is Synthetic Lethal in Mesotheliomas Harboring MTAP Loss: Topic: Mesothelioma Transitional

Mesothelioma remains an incurable cancer with limited therapy. Genetically targeted personalized treatment strategies are currently lacking. Mesotheliomas harbor frequent loss of chromosome 9p21.3 locus encoding for CDKN2A and frequently encompassing methyladenosine phosphoryl transferase (MTAP). Loss of MTAP has recently been shown to be associated with dependency on the symmetrical demethylation of arginine-4 on histone H4 methyltransferase PRMT5. We sought to determine whether mesothelioma cells with MTAP HD would be vulnerable to inhibition of PRMT5, and to explore the pharmacodynamics associated with its suppression. Genome-wide copy number variation (CNV) analysis was undertaken in 94 patients. CNVs were determined using the array based platform, Affymetrix Oncoscan v3. Multiregional whole exome sequencing was also performed on samples from 6 patients. The expression of MTAP and the effect of the drugs tested on H4R3Me2s was evaluated by western blot. Cell growth was analyzed by clonogenic assay after focused RNAi targeting MTAP and/or PRMT5, or treatment with a PRMT5 inhibitor. Oncoscan analysis identified homozygous loss of CDKN2A in 50%, and heterozygous loss in 20.2% of patients. Homozygous loss of MTAP was seen in 39.3% and heterozygous loss in 28.7%, 76.6% of patients had concurrent loss of CDKN2A and MTAP. Homozygous MTAP deletion was found to be present in all regions of tumor ie a truncal deletion, in 3 out of 6 patients. In 65 patients treated with surgery only, homozygous loss of CDKN2A or MTAP was prognostic for progression free survival (CDKN2A: 7.5 vs. 32.9 months, HR 4.536 95% CI 1.765-11.659, p=0.002; MTAP: 7.5 vs. 18.4 months, HR 3.289 95% CI 1.314-8.237, p=0.007). To determine the dependency of MTAP negative cells on PRMT5, we used either siRNA targeting PRMT5 or a PRMT5 inhibitor. PRMT5 silencing did not induce measurable apoptosis however a significant suppression of clonogenic activity was observed after 10 days in MTAP negative cells (H2591). The same effect was observed after concurrent silencing of PRMT5 and MTAP in MTAP positive mesothelioma cells (MPP89). We then utilized a PRMT5 small molecule inhibitor, EPZ015666, to recapitulate RNAi knockdown. Although a clear suppression of H4R3Me2s was observed after 96 hours treatment, the relative potency on clonogenic assay was less than RNAi. Homozygous deletion of MTAP is a common genetic event in mesothelioma. Suppression of PRMT5 represents a novel potential approach for targeting mesotheliomas with 9p21.3 loss. The discrepancy between small molecule inhibitors and RNAi, suggests that PRMT5 dependence may require functions that extend beyond its methyltransferase function.

Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme’s conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5–24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme’s fold.

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the (18)O KIEs on the 2'O nucleophile, 5'O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5'O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5'O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2'-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.

Structure of the Legionella effector AnkX reveals the mechanism of phosphocholine transfer by the FIC domain

The FIC motif and the eukaryotic-like ankyrin repeats are found in many bacterial type IV effectors, yet little is known about how these domains enable bacteria to modulate host cell functions. Bacterial FIC domains typically bind ATP and transfer adenosine monophosphate moiety onto target proteins. The ankyrin repeat-containing protein AnkX encoded by the intracellular pathogen Legionella pneumophila is unique in that its FIC domain binds to CDP-choline and transfers a phosphocholine residue onto proteins in the Rab1 GTPase family. By determining the structures of unbound AnkX and AnkX with bound CDP-choline, CMP/phosphocholine and CMP, we demonstrate that the orientation of substrate binding in relation to the catalytic FIC motif enables this protein to function as a phosphocholinating enzyme rather than a nucleotidyl transferase. Additionally, the structure reveals that the ankyrin repeats mediate scaffolding interactions that resemble those found in protein-protein interactions, but are unprecedented in intramolecular interactions. Together with phosphocholination experiments, our structures unify a general phosphoryl transferase mechanism common to all FIC enzymes that should be conserved from bacteria to human.

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy

Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation.

Structures of Staphylococcus aureus D-Tagatose-6-phosphate Kinase Implicate Domain Motions in Specificity and Mechanism

High resolution structures of Staphylococcus aureus d-tagatose-6-phosphate kinase (LacC) in two crystal forms are herein reported. The structures define LacC in apoform, in binary complexes with ADP or the co-factor analogue AMP-PNP, and in a ternary complex with AMP-PNP and D-tagatose-6-phosphate. The tertiary structure of the LacC monomer, which is closely related to other members of the pfkB subfamily of carbohydrate kinases, is composed of a large alpha/beta core domain and a smaller, largely beta "lid." Four extended polypeptide segments connect these two domains. Dimerization of LacC occurs via interactions between lid domains, which come together to form a beta-clasp structure. Residues from both subunits contribute to substrate binding. LacC adopts a closed structure required for phosphoryl transfer only when both substrate and co-factor are bound. A reaction mechanism similar to that used by other phosphoryl transferases is proposed, although unusually, when both substrate and co-factor are bound to the enzyme two Mg(2+) ions are observed in the active site. A new motif of amino acid sequence conservation common to the pfkB subfamily of carbohydrate kinases is identified.

Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily.

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

The tetrahymena ribozyme cleaves a 5'-methylene phosphonate monoester approximately 10(2)-fold faster than a normal phosphate diester: implications for enzyme catalysis of phosphoryl transfer reactions.

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by <5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.

Inositol phosphoryl transferases from human pathogenic fungi

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A ( AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.

Interferon therapy of hematologic malignancies.

Chronic myelogenous leukemia (CML) is a hematologic neoplasm characterized by the proliferation and accumulation of mature myeloid cells and their progenitors. It is a clonal disorder caused by somatic mutation in a pluripotent hematopoietic stem cell; consequently, there is involvement of myeloid and erythroid cells, monocytes/macrophages, megakaryocytes, and lymphocytes. Clonality has been conclusively demonstrated by polymorphic genetic systems such as glucose 6-phosphate dehydrogenase (G6PD) in heterozygous individuals with CML, X-chromosome DNA restriction fragment-length polymorphism of hypoxanthine glucuronyl ribosyl phosphoryl transferase, and the presence of the Philadelphia (Ph’) chromosome and other clonal cytogenetic markers [1-5].

Aspartic acid 252 and asparagine 185 are essential for activity of lipid N-acetylglucosaminylphosphate transferase.

A key step in the assembly of oligosaccharide-lipid intermediates in N-linked glycosylation is the transfer of N-acetylglucosamine 1-phosphate to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosaminyl:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT). Comparison of the amino acid sequences of L-G1PT from five diverse species showed 75 amino acids identical in all five proteins. Using site-directed mutagenesis, we analyzed the importance of a number of these conserved residues to the enzymatic activity of L-G1PT using a plasmid shuffling procedure in Schizosaccharomyces pombe. S. pombe cells containing a chromosomal deletion of the essential gpt+ gene are rescued by a plasmid containing the S. pombe gpt open reading frame. Replacement of that plasmid by a plasmid encoding a mutated hamster L-G1PT cDNA sequence indicated that the mutated protein provided sufficient enzyme activity to permit cell growth. Mutations of aspartic acid 252 and asparagine 185 did not allow plasmid shuffling, indicating these residues were essential for activity. A combination of mutations at asparagine 182 and tryptophan 122 did not allow plasmid shuffling, although the single mutations did. Overexpression of the mutant proteins in S. pombe conferred tunicamycin (TM) resistance, indicating that the mutant proteins had a conformation necessary for binding TM, a substrate analog. The mutant proteins were also detected in Western blots and were correctly localized to the membrane fractions. However, the overexpressed proteins did not increase the endogenous level of enzymatic activity in these cells, indicating they were enzymatically inactive.

A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates.

Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli. The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F. Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors. Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F. The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993). The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.

Genomic organization and expression of hamster UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase.

The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT) was found to extend over 6.5 kb and to contain nine exons. The exons ranged in size from 63 to 214 bp, encoding the 408 amino acid protein. The introns ranged from 85 bp to 1.4 kb. Upstream 5' sequences included two possible TATA boxes, one possible CCAAT box and at least two potential GC boxes. Heterologous expression was successful in Schizosaccharomyces pombe, and resulted in cells that were tunicamycin resistant and had 12-fold more L-G1PT activity than wild-type cells. Antiserum prepared to a hydrophilic peptide (residues 300-320) of the L-G1PT protein reacted with a 35-36 kDa protein in membrane samples from Chinese hamster ovary (CHO) cells and S. pombe cells that had increased levels of L-G1PT activity. In both cases, antigenic peptide competed with the 35-36 kDa protein detected by the antiserum.

Partial purification and characterization of a soluble acid phosphatase from the tapeworm,Hymenolepis diminuta

An acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adult Hymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130,000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44 degrees C and pH 5.0. The enzyme appeared to be a monomer with a molecular weight of approximately 62,000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters, and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4 and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.

Spreading of streptomycin antibiotic resistance gene in Escherichia coli plasmids demonstrated by Southern blot analysis.

Plasmids from E. coli strains of 38 donors were transconjugated to common recipient SY663 Escherichia coli K12. The restriction patterns of the isolated plasmids were highly heterogenous. However, the streptomycin (Sm) resistance genes of the plasmids were identical or closely homologous in 29 of the 33 plasmids conferring Sm resistance. These data were based on Southern blot analysis, using the Sm resistance gene (encoding aminoglycoside phosphoryl transferase) as probe cut out from pBP1 plasmid. Our data suggest an extensive spreading of streptomycin resistance gene of this type.

Studies on the mechanism of action of phosphoryl transferase enzymes.

Studies on the mechanism of action of phosphoryl transferase enzymes.

Nucleoside-triphosphatase and hydrolysis of thiamin triphosphate in Escherichia coli.

A membrane-bound nonspecific triphosphatase of E. coli was solubilized and purified to a homogeneous SDS-acrylamide gel electrophoresis band. It was found to be a single polypeptide of 16 kDa requiring no Mg2+, with an optimal pH at 6.5. The substrate specificity was broad and a nonspecific Mg2+-independent ribonucleoside-triphosphatase (NTPase) activity was expressed together with thiamin-triphosphatase activity. The molecular size and characteristics were clearly different from the known NTPase (EC 3.6.1.15). Using the purified thiamin-triphosphatase II, ATP:thiamin-diphosphate phosphoryl transferase (EC 2.7.4.15) activity was demonstrated with an optimal pH of approx. 5.3. Considering its kinetic parameters and other characteristics, however, the thiamin triphosphate synthesizing activity was not thought to take part in cellular thiamin triphosphate synthesis. The possibility that thiamin-triphosphatase II plays a part in the hydrolysis of thiamin triphosphate to control its cellular level is suggested.

Leigh's disease in an adult with evidence of “inhibitor factor” in family members

A 21-year-old man with a longstanding history of impaired visual acuity, strabismus, broad-based gait, and below-average intellectual capacity developed respiratory difficulties and intermittent generalized weakness at age 19. He subsequently showed signs of massive brainstem dysfunction and died. Postmortem examination demonstrated changes compatible with subacute necrotizing encephalomyelopathy of Leigh involving portions of the diencephalon, midbrain, pons, and medulla as well as portions of the spinal cord and optic nerves. Evaluation of family members for presence of the urinary inhibitor factor for thiamine diphosphate phosphoryl transferase revealed abnormal levels in a brother, a maternal uncle, and the maternal grandfather of the patient.