Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing.

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (∼530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (α) located near a 5' splice site, which greatly increases use of this 5' splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

Role of thiamine pyrophosphate in oligomerisation, functioning and import of peroxisomal 2-hydroxyacyl-CoA lyase.

During peroxisomal α-oxidation, the CoA-esters of phytanic acid and 2-hydroxylated straight chain fatty acids are cleaved into a (n-1) fatty aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase (HACL1). HACL1 is imported into peroxisomes via the PEX5/PTS1 pathway, and so far, it is the only known peroxisomal TPP-dependent enzyme in mammals. In this study, the effect of mutations in the TPP-binding domain of HACL1 on enzyme activity, subcellular localisation and oligomerisation was investigated. Mutations of the aspartate 455 and serine 456 residues within the TPP binding domain of the human HACL1 did not affect the targeting upon expression in transfected CHO cells, although enzyme activity was abolished. Gel filtration of native and mutated N-His(6)-fusions, expressed in yeast, revealed that the mutations did not influence the oligomerisation of the (apo)enzyme. Subcellular fractionation of yeast cells expressing HACL1 showed that the lyase activity sedimented at high density in a Nycodenz gradient. In these fractions TPP could be measured, but not when mutated HACL1 was expressed, although the recombinant enzyme was still targeted to peroxisomes. These findings indicate that the binding of TPP is not required for peroxisomal targeting and correct folding of HACL1, in contrast to other TPP-dependent enzymes, and suggest that transport of TPP into peroxisomes is dependent on HACL1 import, without requirement of a specific solute transporter.

Flipping off the riboswitch: RNA structures that control gene expression.

Riboswitches are metabolite-sensing RNA structures that have been discovered in regulatory regions of messenger RNA (mRNA). They have the remarkable ability to shut off the transcription or translation of their own mRNAs in response to binding a specific metabolite. In other words, riboswitches regulate their own genes using RNA instead of protein. Three new crystal structures reveal how S-adenosylmethionine and thiamine pyrophosphate riboswitches accomplish this task.

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate.

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase.

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Mutation thi81 causing a deficiency in the signal transduction of thiamine pyrophosphate in Saccharomyces cerevisiae.

We isolated a strain carrying a recessive constitutive mutation (thi81) for the expression of thiamine metabolism in Saccharomyces cerevisiae. The thi81 mutant exhibits significant thiamine transport, thiamine-repressible acid phosphatase (T-rAPase) activities and significant activities of enzymes involved in thiamine biosynthesis which are repressed in the wild-type strain in medium supplemented with thiamine (2 x 10(-7) M). The thi81 mutant exhibited the same level of thiamine pyrophosphokinase activity and intracellular thiamine pyrophosphate concentration as the wild-type strain in medium supplemented with exogenous thiamine. The mutant strain constitutively produced PHO3 mRNA encoding T-rAPase in medium supplemented with thiamine. These results suggest that the thi81 mutant lacks a negative factor involved in the regulation of the genes encoding proteins involved in yeast thiamine metabolism.

A non-cofactor role of thiamine derivatives in excitable cells?

Thiamine diphosphate (TDP) is an important cofactor of pyruvate (PDH) and alpha-ketoglutarate (KGDH) dehydrogenases and transketolase. Thiamine deficiency leads to reversible and irreversible brain lesions due to impaired oxidative metabolism. A specific non-cofactor role for thiamine has also been proposed in excitable cells and thiamine triphosphate (TTP) might be involved in the regulation of ion channels. Thiamine is taken up by neuroblastoma cells through a high affinity transporter. Inside the cells, it is rapidly phosphorylated to TDP. This high turnover TDP pool is the precursor for TTP. Most of the TDP however has a low turnover and is associated with PDH and KGDH in mitochondria. In excised inside-out patches from neuroblastoma cells, TTP, at a concentration of 1 microM, activates chloride channels of large unitary conductance, the so-called maxi-Cl- channels. These channels are inhibited by oxythiamine from the outide. In addition to the role of TTP in the regulation of chloride channels, thiamine itself, or a presently unknown analog, may have trophic effects on neuronal cells.

The thiamine-dependent hysteretic behavior of human transketolase: implications for thiamine deficiency.

We have investigated the hysteretic properties of human transketolase with emphasis on its dependency on thiamine pyrophosphate concentration. As demonstrated previously, the reaction progress curves revealed a slow transition from an initial low velocity to a faster final steady-state velocity, characterized by the rate constant tau-1. The rate of the transition was dependent on the concentration of the thiamine pyrophosphate cofactor, with progressively longer transition times found as the concentration of thiamine pyrophosphate was decreased. At physiological thiamine pyrophosphate concentrations, the inverse rate constant was in the range of 10 to 20 min for fibroblast-derived transketolase and increased dramatically with only small decreases from these levels of thiamine pyrophosphate. Variation in the lag was found when transketolase from different individuals was examined. Moreover, at low levels of thiamine, the rate of the transition was different between fibroblast- and lymphoblast-derived transketolase. The substantial lag in formation of active holoenzyme and the findings of interindividual variation and cell type variation in the lag period suggest mechanisms for the loss of transketolase activity during thiamine deficiency and may explain, at least in part, the differential sensitivity to deficiency demonstrated by tissues and individuals.

A common structural motif in thiamin pyrophosphate-binding enzymes.

The amino acid sequences of a wide range of enzymes that utilize thiamin pyrophosphate (TPP) as cofactor have been compared. A common sequence motif approximately 30 residues in length was detected, beginning with the highly conserved sequence -GDG- and concluding with the highly conserved sequence -NN-. Secondary structure predictions suggest that the motif may adopt a beta alpha beta fold. The same motif was recognised in the primary structure of a protein deduced from the DNA sequence of a hitherto unassigned open reading frame of Rhodobacter capsulata. This putative protein exhibits additional homology with some but not all of the TPP-binding enzymes.

[Lag period as affected by the adrenal pyruvate dehydrogenase complex containing residual endogenous thiamine pyrophosphate]. / Lag-period pri funktsionirovanii piruvatdegidrogenaznogo kompleksa nadpochechnikov, soderzhashchego ostatochnyi éndogennyi tiaminpirofosfat.

The pyruvate dehydrogenase complex from bovine adrenals does not completely lose thiamine pyrophosphate (TPP) in the course of purification. The preparations contain firmly bound residual TPP in the amount providing for one third of the maximal activity of the complex. A lag period (tau) is observed during catalysis by the pyruvate dehydrogenase complex with the residual TPP in the absence of exogenous coenzyme. The duration of the lag period depends on concentration of the complex: tau is decreased as the concentration increases. The decrease of the lag period occurs after preincubation of the complex with pyruvate, but it is the most remarkable after preincubation with pyruvate and CoA. The tetrahydrothiamine pyrophosphate at low concentrations completely inhibits the activity. However, the residual TPP is retained as is evidenced by the recovery of the complex activity after gel-filtration through Sephadex G-25. The development of the lag period may be interpreted in terms of partial limited dissociation of the residual TPP in the region of the catalytic center.

Possible role of thiamine in neuromuscular transmission.

Pyrithiamine (50 mg/kg), a thiamine antagonist, decreased the muscle twitches of the rat masseter muscle at stimulation frequencies above 1 Hz 40--80 min after an i.v. injection. The post-tetanic potentiation (PTP) induced by nerve stimulation of the masseter muscle was abolished by pyrithiamine. Administration of thiamine restored the muscle twitches at stimulation frequencies above 1 Hz and the PTP. The muscle twitches elicited by direct muscle stimulation were not affected by pyrithiamine treatment. The abolishment of the PTP was accompanied by a decrease in thiamine and thiamine-diphosphate. The pyruvate level in the blood was unchanged after pyrithiamine treatment. Oxythiamine, on the other hand, had no effect on the PTP but increased the pyruvate level in the blood. Fern extract which contains thiaminase I also abolished the PTP--an effect reversible by the addition of thiamine. The frequency-induced depression of the muscle twitches induced by pyrithiamine was similar to the effect of low doses of d-tubocurarine (8 microgram/kg). The results support the hypothesis that thiamine may play a role in neuromuscular transmission.

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.